|
1
|
Chadda A, Nguyen B, Lohman TM, Galburt EA. Structural basis for dimerization and activation of UvrD-family helicases. Proc Natl Acad Sci U S A 2025; 122:e2422330122. [PMID: 40048277 PMCID: PMC11912403 DOI: 10.1073/pnas.2422330122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2024] [Accepted: 02/07/2025] [Indexed: 03/12/2025] Open
Abstract
UvrD-family helicases are superfamily 1A motor proteins that function during DNA replication, recombination, repair, and transcription. UvrD family monomers translocate along single-stranded (ss) DNA but need to be activated by dimerization to unwind DNA in the absence of force or accessory factors. However, prior structural studies have only revealed monomeric complexes. Here, we report the first structures of a dimeric UvrD-family helicase, Mycobacterium tuberculosis UvrD1, both free and bound to a DNA junction. In each structure, the dimer interface occurs between the 2B subdomains of each subunit. The apo UvrD1 dimer is observed in symmetric compact and extended forms indicating substantial flexibility. This symmetry is broken in the DNA-bound dimer complex with leading and trailing subunits adopting distinct conformations. Biochemical experiments reveal that the Escherichia coli UvrD dimer shares the same 2B-2B interface. In contrast to the dimeric structures, an inactive, autoinhibited UvrD1 DNA-bound monomer structure reveals 2B subdomain-DNA contacts that are likely inhibitory. The major reorientation of the 2B subdomains that occurs upon UvrD1 dimerization prevents these duplex DNA interactions, thus relieving the autoinhibition. These structures reveal that the 2B subdomain serves a major regulatory role rather than participating directly in DNA unwinding.
Collapse
Affiliation(s)
- Ankita Chadda
- Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis School of Medicine, Saint Louis, MO63110
| | - Binh Nguyen
- Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis School of Medicine, Saint Louis, MO63110
| | - Timothy M. Lohman
- Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis School of Medicine, Saint Louis, MO63110
| | - Eric A. Galburt
- Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis School of Medicine, Saint Louis, MO63110
| |
Collapse
|
|
2
|
Dai J, Liu R, He S, Li T, Hu Y, Huang H, Li Y, Guo X. The Role of SF1 and SF2 Helicases in Biotechnological Applications. Appl Biochem Biotechnol 2024; 196:9064-9084. [PMID: 39093351 DOI: 10.1007/s12010-024-05027-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/23/2024] [Indexed: 08/04/2024]
Abstract
Helicases, which utilize ATP hydrolysis to separate nucleic acid duplexes, play crucial roles in DNA and RNA replication, repair, recombination, and transcription. Categorized into the major groups superfamily 1 (SF1) and superfamily 2 (SF2), alongside four minor groups, these proteins exhibit a conserved catalytic core indicative of a shared evolutionary origin while displaying functional diversity through interactions with various substrates. This review summarizes the structures, functions and mechanisms of SF1 and SF2 helicases, with an emphasis on conserved ATPase sites and RecA-like domains essential for their enzymatic and nucleic acid binding capabilities. It highlights the unique 1B and 2B domains in SF1 helicases and their impact on enzymatic activity. The DNA unwinding process is detailed, covering substrate recognition, ATP hydrolysis, and conformational changes, while addressing debates over the active form of UvrD helicase and post-unwinding dissociation. More importantly, this review discusses the biotechnological potential of helicases in emerging technologies such as nanopore sequencing, protein sequencing, and isothermal amplification, focusing on their use in pathogen detection, biosensor enhancement, and cancer treatment. As understanding deepens, innovative applications in genome editing, DNA sequencing, and synthetic biology are anticipated.
Collapse
Affiliation(s)
- Jing Dai
- Dongguan Key Laboratory of Public Health Laboratory Science, School of Public Health, Guangdong Medical University, Dongguan, 523808, People's Republic of China
| | - Ronghui Liu
- School of Microelectronic, Southern University of Science and Technology, Shenzhen, 518000, People's Republic of China.
| | - Shujun He
- Dongguan Key Laboratory of Public Health Laboratory Science, School of Public Health, Guangdong Medical University, Dongguan, 523808, People's Republic of China
| | - Tie Li
- School of Microelectronic, Southern University of Science and Technology, Shenzhen, 518000, People's Republic of China
| | - Yuhang Hu
- School of Microelectronic, Southern University of Science and Technology, Shenzhen, 518000, People's Republic of China
| | - Huiqun Huang
- Dongguan Key Laboratory of Public Health Laboratory Science, School of Public Health, Guangdong Medical University, Dongguan, 523808, People's Republic of China
| | - Yi Li
- School of Microelectronic, Southern University of Science and Technology, Shenzhen, 518000, People's Republic of China.
| | - Xinrong Guo
- Dongguan Key Laboratory of Public Health Laboratory Science, School of Public Health, Guangdong Medical University, Dongguan, 523808, People's Republic of China.
| |
Collapse
|
|
3
|
Diaz Escarcega R, Marshall P, Tsvetkov AS. G-quadruplex DNA and RNA in cellular senescence. FRONTIERS IN AGING 2024; 5:1491389. [PMID: 39444378 PMCID: PMC11496277 DOI: 10.3389/fragi.2024.1491389] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/04/2024] [Accepted: 09/25/2024] [Indexed: 10/25/2024]
Abstract
Normal cells divide, are damaged, and are repaired across their lifetime. As cells age, they enter cellular senescence, characterized by a permanent state of cell-cycle arrest triggered by various stressors. The molecular mechanisms that regulate senescent phenotypes have been actively investigated over the last several decades; however, one area that has been neglected is how G-quadruplex (G4) DNA and RNA (G4-DNA and G4-RNA) mediate senescence. These non-canonical four-stranded DNA and RNA structures regulate most normative DNA and RNA-dependent processes, such as transcription, replication, and translation, as well as pathogenic mechanisms, including genomic instability and abnormal stress granule function. This review also highlights the contribution of G4s to sex differences in age-associated diseases and emphasizes potential translational approaches to target senescence and anti-aging mechanisms through G4 manipulation.
Collapse
Affiliation(s)
- Rocio Diaz Escarcega
- Department of Neurology, University of Texas McGovern Medical School, Houston, TX, United States
| | - Paul Marshall
- College of Health and Medicine, The Australian National University, Canberra, ACT, Australia
| | - Andrey S. Tsvetkov
- Department of Neurology, University of Texas McGovern Medical School, Houston, TX, United States
- The University of Texas Graduate School of Biomedical Sciences, Houston, TX, United States
- UTHealth Consortium on Aging, The University of Texas McGovern Medical School, Houston, TX, United States
| |
Collapse
|
|
4
|
Chadda A, Nguyen B, Lohman TM, Galburt EA. Structural Basis for Dimerization and Activation of UvrD-family Helicases. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.09.05.611425. [PMID: 39282289 PMCID: PMC11398504 DOI: 10.1101/2024.09.05.611425] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 10/21/2024]
Abstract
UvrD-family helicases are superfamily 1A motor proteins that function during DNA replication, recombination, repair, and transcription. UvrD family monomers translocate along single stranded (ss) DNA but need to be activated by dimerization to unwind DNA in the absence of force or accessory factors. However, prior structural studies have only revealed monomeric complexes. Here, we report the first structures of a dimeric UvrD-family helicase, Mycobacterium tuberculosis UvrD1, both free and bound to a DNA junction. In each structure, the dimer interface occurs between the 2B subdomains of each subunit. The apo UvrD1 dimer is observed in symmetric compact and extended forms indicating substantial flexibility. This symmetry is broken in the DNA-bound dimer complex with leading and trailing subunits adopting distinct conformations. Biochemical experiments reveal that the E. coli UvrD dimer shares the same 2B-2B interface. In contrast to the dimeric structures, an inactive, auto-inhibited UvrD1 DNA-bound monomer structure reveals 2B subdomain-DNA contacts that are likely inhibitory. The major re-orientation of the 2B subdomains that occurs upon UvrD1 dimerization prevents these duplex DNA interactions, thus relieving the auto-inhibition. These structures reveal that the 2B subdomain serves a major regulatory role rather than participating directly in DNA unwinding.
Collapse
Affiliation(s)
- Ankita Chadda
- Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis School of Medicine, Saint Louis, MO 63110
| | - Binh Nguyen
- Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis School of Medicine, Saint Louis, MO 63110
| | - Timothy M Lohman
- Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis School of Medicine, Saint Louis, MO 63110
| | - Eric A Galburt
- Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis School of Medicine, Saint Louis, MO 63110
| |
Collapse
|
|
5
|
Lei Y, Onaka H, Asamizu S. Transcriptionally induced nucleoid-associated protein-like ccr1 in combined-culture serves as a global effector of Streptomyces secondary metabolism. Front Microbiol 2024; 15:1422977. [PMID: 39070263 PMCID: PMC11272600 DOI: 10.3389/fmicb.2024.1422977] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2024] [Accepted: 06/26/2024] [Indexed: 07/30/2024] Open
Abstract
Combined-cultures involving mycolic acid-containing bacteria (MACB) can stimulate secondary metabolite (SM) production in actinomycetes. In a prior investigation, we screened Streptomyces coelicolor JCM4020 mutants with diminished production of SMs, specifically undecylprodigiosin (RED), which was enhanced by introducing the MACB Tsukamurella pulmonis TP-B0596. In this study, we conducted mutational analysis that pinpointed the sco1842 gene, which we assigned the gene name ccr1 (combined-culture related regulatory protein no. 1), as a crucial factor in the deficient phenotype observed in the production of various major SMs in S. coelicolor A3(2). Notably, the Ccr1 (SCO1842) homolog was found to be highly conserved throughout the Streptomyces genome. Although Ccr1 lacked conserved motifs, in-depth examination revealed the presence of a helix-turn-helix (HTH) motif in the N-terminal region and a helicase C-terminal domain (HCTD) motif in the C-terminal region in some of its homologs. Ccr1 was predicted to be a nucleoid-associated protein (NAP), and its impact on gene transcription was validated by RNA-seq analysis that revealed genome-wide variations. Furthermore, RT-qPCR demonstrated that ccr1 was transcriptionally activated in combined-culture with T. pulmonis, which indicated that Ccr1 is involved in the response to bacterial interaction. We then investigated Streptomyces nigrescens HEK616 in combined-culture, and the knockout mutant of the ccr1 homolog displayed reduced production of streptoaminals and 5aTHQs. This finding reveals that the Ccr1 homolog in Streptomyces species is associated with SM production. Our study elucidates the existence of a new family of NAP-like proteins that evolved in Streptomyces species and play a pivotal role in SM production.
Collapse
Affiliation(s)
- Yukun Lei
- Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Hiroyasu Onaka
- Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
- Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan
- Department of Life Science, Faculty of Science, Gakushuin University, Tokyo, Japan
| | - Shumpei Asamizu
- Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
- Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan
- Engineering Biology Research Center, Kobe University, Kobe, Japan
| |
Collapse
|
|
6
|
Nolte DD. Coherent light scattering from cellular dynamics in living tissues. REPORTS ON PROGRESS IN PHYSICS. PHYSICAL SOCIETY (GREAT BRITAIN) 2024; 87:036601. [PMID: 38433567 DOI: 10.1088/1361-6633/ad2229] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/01/2022] [Accepted: 01/24/2024] [Indexed: 03/05/2024]
Abstract
This review examines the biological physics of intracellular transport probed by the coherent optics of dynamic light scattering from optically thick living tissues. Cells and their constituents are in constant motion, composed of a broad range of speeds spanning many orders of magnitude that reflect the wide array of functions and mechanisms that maintain cellular health. From the organelle scale of tens of nanometers and upward in size, the motion inside living tissue is actively driven rather than thermal, propelled by the hydrolysis of bioenergetic molecules and the forces of molecular motors. Active transport can mimic the random walks of thermal Brownian motion, but mean-squared displacements are far from thermal equilibrium and can display anomalous diffusion through Lévy or fractional Brownian walks. Despite the average isotropic three-dimensional environment of cells and tissues, active cellular or intracellular transport of single light-scattering objects is often pseudo-one-dimensional, for instance as organelle displacement persists along cytoskeletal tracks or as membranes displace along the normal to cell surfaces, albeit isotropically oriented in three dimensions. Coherent light scattering is a natural tool to characterize such tissue dynamics because persistent directed transport induces Doppler shifts in the scattered light. The many frequency-shifted partial waves from the complex and dynamic media interfere to produce dynamic speckle that reveals tissue-scale processes through speckle contrast imaging and fluctuation spectroscopy. Low-coherence interferometry, dynamic optical coherence tomography, diffusing-wave spectroscopy, diffuse-correlation spectroscopy, differential dynamic microscopy and digital holography offer coherent detection methods that shed light on intracellular processes. In health-care applications, altered states of cellular health and disease display altered cellular motions that imprint on the statistical fluctuations of the scattered light. For instance, the efficacy of medical therapeutics can be monitored by measuring the changes they induce in the Doppler spectra of livingex vivocancer biopsies.
Collapse
Affiliation(s)
- David D Nolte
- Department of Physics and Astronomy, Purdue University, West Lafayette, IN 47907, United States of America
| |
Collapse
|
|
7
|
Fazio NT, Mersch KN, Hao L, Lohman TM. E. coli RecB Nuclease Domain Regulates RecBCD Helicase Activity but not Single Stranded DNA Translocase Activity. J Mol Biol 2024; 436:168381. [PMID: 38081382 PMCID: PMC11131135 DOI: 10.1016/j.jmb.2023.168381] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Revised: 11/30/2023] [Accepted: 12/01/2023] [Indexed: 12/23/2023]
Abstract
Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as occurring by the ATPase motors mechanically pulling the DNA duplex across a wedge domain in the helicase, biochemical data show that processive DNA unwinding by E. coli RecBCD helicase can occur in the absence of ssDNA translocation by the canonical RecB and RecD motors. Here we show that DNA unwinding is not a simple consequence of ssDNA translocation by the motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecBΔNucCD) unwinds dsDNA at significantly slower rates than RecBCD, while the ssDNA translocation rate is unaffected. This effect is primarily due to the absence of the nuclease domain since a nuclease-dead mutant (RecBD1080ACD), which retains the nuclease domain, showed no change in ssDNA translocation or dsDNA unwinding rates relative to RecBCD on short DNA substrates (≤60 base pairs). Hence, ssDNA translocation is not rate-limiting for DNA unwinding. RecBΔNucCD also initiates unwinding much slower than RecBCD from a blunt-ended DNA. RecBΔNucCD also unwinds DNA ∼two-fold slower than RecBCD on long DNA (∼20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecBD1080ACD unwinding are intermediate between RecBCD and RecBΔNucCD. Surprisingly, significant pauses in DNA unwinding occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, possibly allosterically and that RecBΔNucCD may mimic a post-chi state of RecBCD.
Collapse
Affiliation(s)
- Nicole T Fazio
- Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, United States
| | - Kacey N Mersch
- Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, United States
| | - Linxuan Hao
- Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, United States
| | - Timothy M Lohman
- Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, United States.
| |
Collapse
|
|
8
|
Dabral P, Uppal T, Verma SC. G-quadruplexes of KSHV oriLyt play important roles in promoting lytic DNA replication. Microbiol Spectr 2023; 11:e0531622. [PMID: 37800915 PMCID: PMC10714766 DOI: 10.1128/spectrum.05316-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2022] [Accepted: 08/15/2023] [Indexed: 10/07/2023] Open
Abstract
IMPORTANCE Biological processes originating from the DNA and RNA can be regulated by the secondary structures present in the stretch of nucleic acids, and the G-quadruplexes are shown to regulate transcription, translation, and replication. In this study, we identified the presence of multiple G-quadruplex sites in the region (oriLyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) DNA, which is essential for DNA replication during the lytic cycle. We demonstrated the roles of these G-quadruplexes through multiple biochemical and biophysical assays in controlling replication and efficient virus production. We demonstrated that KSHV achieves this by recruiting RecQ1 (helicase) at those G-quadruplex sites for efficient viral DNA replication. Analysis of the replicated DNA through nucleoside labeling and immunostaining showed a reduced initiation of DNA replication in cells with a pharmacologic stabilizer of G-quadruplexes. Overall, this study confirmed the role of the G-quadruplex in regulating viral DNA replication, which can be exploited for controlling viral DNA replication.
Collapse
Affiliation(s)
- Prerna Dabral
- Department of Microbiology and Immunology, University of Nevada, Reno School of Medicine, Reno, Nevada, USA
- Vitalant Research Institute, San Francisco, California, USA
| | - Timsy Uppal
- Department of Microbiology and Immunology, University of Nevada, Reno School of Medicine, Reno, Nevada, USA
| | - Subhash C. Verma
- Department of Microbiology and Immunology, University of Nevada, Reno School of Medicine, Reno, Nevada, USA
| |
Collapse
|
|
9
|
Oh H, Koo J, An SY, Hong SH, Suh JY, Bae E. Structural and functional investigation of GajB protein in Gabija anti-phage defense. Nucleic Acids Res 2023; 51:11941-11951. [PMID: 37897358 PMCID: PMC10681800 DOI: 10.1093/nar/gkad951] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Revised: 09/13/2023] [Accepted: 10/12/2023] [Indexed: 10/30/2023] Open
Abstract
Bacteriophages (phages) are viruses that infect bacteria and archaea. To fend off invading phages, the hosts have evolved a variety of anti-phage defense mechanisms. Gabija is one of the most abundant prokaryotic antiviral systems and consists of two proteins, GajA and GajB. GajA has been characterized experimentally as a sequence-specific DNA endonuclease. Although GajB was previously predicted to be a UvrD-like helicase, its function is unclear. Here, we report the results of structural and functional analyses of GajB. The crystal structure of GajB revealed a UvrD-like domain architecture, including two RecA-like core and two accessory subdomains. However, local structural elements that are important for the helicase function of UvrD are not conserved in GajB. In functional assays, GajB did not unwind or bind various types of DNA substrates. We demonstrated that GajB interacts with GajA to form a heterooctameric Gabija complex, but GajB did not exhibit helicase activity when bound to GajA. These results advance our understanding of the molecular mechanism underlying Gabija anti-phage defense and highlight the role of GajB as a component of a multi-subunit antiviral complex in bacteria.
Collapse
Affiliation(s)
- Hyejin Oh
- Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Korea
| | - Jasung Koo
- Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Korea
| | - So Young An
- Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Korea
- Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea
| | - Sung-Hyun Hong
- Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Korea
- Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea
| | - Jeong-Yong Suh
- Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Korea
- Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea
| | - Euiyoung Bae
- Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Korea
- Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea
| |
Collapse
|
|
10
|
Fazio N, Mersch KN, Hao L, Lohman TM. E. coli RecBCD Nuclease Domain Regulates Helicase Activity but not Single Stranded DNA Translocase Activity. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.10.13.561901. [PMID: 37905078 PMCID: PMC10614803 DOI: 10.1101/2023.10.13.561901] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/02/2023]
Abstract
Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as a consequence of mechanically pulling the DNA duplex across a wedge domain in the helicase by the single stranded (ss)DNA translocase activity of the ATPase motors, biochemical data indicate that processive DNA unwinding by the E. coli RecBCD helicase can occur in the absence of ssDNA translocation of the canonical RecB and RecD motors. Here, we present evidence that dsDNA unwinding is not a simple consequence of ssDNA translocation by the RecBCD motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecB ΔNuc CD) unwinds dsDNA at significantly slower rates than RecBCD, while the rate of ssDNA translocation is unaffected. This effect is primarily due to the absence of the nuclease domain and not the absence of the nuclease activity, since a nuclease-dead mutant (RecB D1080A CD), which retains the nuclease domain, showed no significant change in rates of ssDNA translocation or dsDNA unwinding relative to RecBCD on short DNA substrates (≤ 60 base pairs). This indicates that ssDNA translocation is not rate-limiting for DNA unwinding. RecB ΔNuc CD also initiates unwinding much slower than RecBCD from a blunt-ended DNA, although it binds with higher affinity than RecBCD. RecB ΔNuc CD also unwinds DNA ∼two-fold slower than RecBCD on long DNA (∼20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecB D1080A CD unwinding are intermediate between RecBCD and RecB ΔNuc CD. Surprisingly, significant pauses occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, rather than DNA translocation, possibly allosterically. Since the rate of DNA unwinding by RecBCD also slows after it recognizes a chi sequence, RecB ΔNuc CD may mimic a post- chi state of RecBCD.
Collapse
|
|
11
|
Kermagoret A, Bardelang D. The Diversity of Cucurbituril Molecular Switches and Shuttles. Chemistry 2023:e202302114. [PMID: 37725407 DOI: 10.1002/chem.202302114] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Revised: 09/15/2023] [Accepted: 09/18/2023] [Indexed: 09/21/2023]
Abstract
Ring translocation switches and shuttles featuring a macrocycle (or a ring molecule) navigating between two or more stations continue to attract attention. While the vast majority of these systems are developed in organic solvents, the cucurbituril (CB) macrocycles are ideally suited to prepare such systems in water. Indeed, their stability and their relatively high affinity for relevant guest molecules are key attributes toward translating the progresses made in organic solvents, into water. This concept article summarizes the findings, key advances and multiple possibilities offered by CBs toward advanced molecular switches and shuttles in water.
Collapse
|
|
12
|
Incicco JJ, Cababie LA, Sarto C, Adler NS, Amrein F, Mikkelsen E, Arrar M, Kaufman SB. Thermodynamic and mechanistic analysis of the functional properties of dengue virus NS3 helicase. Biophys Rev 2023; 15:591-600. [PMID: 37681085 PMCID: PMC10480135 DOI: 10.1007/s12551-023-01101-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Accepted: 07/17/2023] [Indexed: 09/09/2023] Open
Abstract
The Dengue Virus (DENV) non-structural protein 3 (NS3) is a multi-functional protein critical in the viral life cycle. The DENV NS3 is comprised of a serine protease domain and a helicase domain. The helicase domain itself acts as a molecular motor, either translocating in a unidirectional manner along single-stranded RNA or unwinding double-stranded RNA, processes fueled by the hydrolysis of nucleoside triphosphates. In this brief review, we summarize our contributions and ongoing efforts to uncover the thermodynamic and mechanistic functional properties of the DENV NS3 as an NTPase and helicase.
Collapse
Affiliation(s)
- J. Jeremías Incicco
- Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Universidad de Buenos Aires, CONICET, Junín 956, Caba, 1113 Argentina
- Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Caba, 1113 Argentina
| | - Leila A. Cababie
- Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Universidad de Buenos Aires, CONICET, Junín 956, Caba, 1113 Argentina
- Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Caba, 1113 Argentina
| | - Carolina Sarto
- Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), Universidad de Buenos Aires. CONICET, Intendente Güiraldes 2160, Caba, 1428 Argentina
| | - Natalia S. Adler
- CONICET. Centro de Investigaciones en Bionanociencias (CIBION), Godoy Cruz 2390, Caba, 1425 Argentina
| | - Fernando Amrein
- Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Universidad de Buenos Aires, CONICET, Junín 956, Caba, 1113 Argentina
- Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Caba, 1113 Argentina
| | - Evelyn Mikkelsen
- Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Universidad de Buenos Aires, CONICET, Junín 956, Caba, 1113 Argentina
- Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Caba, 1113 Argentina
| | - Mehrnoosh Arrar
- Instituto de Cálculo, Universidad de Buenos Aires. CONICET, Intendente Güiraldes 2160, Caba, 1428 Argentina
| | - Sergio B. Kaufman
- Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Universidad de Buenos Aires, CONICET, Junín 956, Caba, 1113 Argentina
- Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Caba, 1113 Argentina
| |
Collapse
|
|
13
|
Becker RA, Hub JS. Molecular simulations of DEAH-box helicases reveal control of domain flexibility by ligands: RNA, ATP, ADP, and G-patch proteins. Biol Chem 2023; 404:867-879. [PMID: 37253384 DOI: 10.1515/hsz-2023-0154] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2023] [Accepted: 05/05/2023] [Indexed: 06/01/2023]
Abstract
DEAH-box helicases use the energy from ATP hydrolysis to translocate along RNA strands. They are composed of tandem RecA-like domains and a C-terminal domain connected by flexible linkers, and the activity of several DEAH-box helicases is regulated by cofactors called G-patch proteins. We used all-atom molecular dynamics simulations of the helicases Prp43, Prp22, and DHX15 in various liganded states to investigate how RNA, ADP, ATP, or G-patch proteins influence their conformational dynamics. The simulations suggest that apo helicases are highly flexible, whereas binding of RNA renders the helicases more rigid. ATP and ADP control the stability of the RecA1-RecA2 interface, but they have only a smaller effect on domain flexibility in absence of a RecA1-RecA2 interface. Binding of a G-patch protein to DHX15 imposes a more structured conformational ensemble, characterized by more defined relative domain arrangements and by an increased conformational stability of the RNA tunnel. However, the effect of the G-patch protein on domain dynamics is far more subtle as compared to the effects of RNA or ATP/ADP. The simulations characterize DEAH-box helicase as dynamic machines whose conformational ensembles are strongly defined by the presence of RNA, ATP, or ADP and only fine-tuned by the presence of G-patch proteins.
Collapse
Affiliation(s)
- Robert A Becker
- Theoretical Physics and Center for Biophysics, Saarland University, Campus E2 6, 66123 Saarbrücken, Germany
| | - Jochen S Hub
- Theoretical Physics and Center for Biophysics, Saarland University, Campus E2 6, 66123 Saarbrücken, Germany
| |
Collapse
|
|
14
|
Becker RA, Hub JS. Continuous millisecond conformational cycle of a DEAH box helicase reveals control of domain motions by atomic-scale transitions. Commun Biol 2023; 6:379. [PMID: 37029280 PMCID: PMC10082070 DOI: 10.1038/s42003-023-04751-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Accepted: 03/23/2023] [Indexed: 04/09/2023] Open
Abstract
Helicases are motor enzymes found in every living organism and viruses, where they maintain the stability of the genome and control against false recombination. The DEAH-box helicase Prp43 plays a crucial role in pre-mRNA splicing in unicellular organisms by translocating single-stranded RNA. The molecular mechanisms and conformational transitions of helicases are not understood at the atomic level. We present a complete conformational cycle of RNA translocation by Prp43 in atomic detail based on molecular dynamics simulations. To enable the sampling of such complex transition on the millisecond timescale, we combined two enhanced sampling techniques, namely simulated tempering and adaptive sampling guided by crystallographic data. During RNA translocation, the center-of-mass motions of the RecA-like domains followed the established inchworm model, whereas the domains crawled along the RNA in a caterpillar-like movement, suggesting an inchworm/caterpillar model. However, this crawling required a complex sequence of atomic-scale transitions involving the release of an arginine finger from the ATP pocket, stepping of the hook-loop and hook-turn motifs along the RNA backbone, and several others. These findings highlight that large-scale domain dynamics may be controlled by complex sequences of atomic-scale transitions.
Collapse
Affiliation(s)
- Robert A Becker
- Theoretical Physics and Center for Biophysics, Saarland University, Saarbrücken, Germany
| | - Jochen S Hub
- Theoretical Physics and Center for Biophysics, Saarland University, Saarbrücken, Germany.
| |
Collapse
|
|
15
|
Slocombe L, Winokan M, Al-Khalili J, Sacchi M. Proton transfer during DNA strand separation as a source of mutagenic guanine-cytosine tautomers. Commun Chem 2022; 5:144. [PMID: 36697962 PMCID: PMC9814255 DOI: 10.1038/s42004-022-00760-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2022] [Accepted: 10/14/2022] [Indexed: 11/06/2022] Open
Abstract
Proton transfer between the DNA bases can lead to mutagenic Guanine-Cytosine tautomers. Over the past several decades, a heated debate has emerged over the biological impact of tautomeric forms. Here, we determine that the energy required for generating tautomers radically changes during the separation of double-stranded DNA. Density Functional Theory calculations indicate that the double proton transfer in Guanine-Cytosine follows a sequential, step-like mechanism where the reaction barrier increases quasi-linearly with strand separation. These results point to increased stability of the tautomer when the DNA strands unzip as they enter the helicase, effectively trapping the tautomer population. In addition, molecular dynamics simulations indicate that the relevant strand separation time is two orders of magnitude quicker than previously thought. Our results demonstrate that the unwinding of DNA by the helicase could simultaneously slow the formation but significantly enhance the stability of tautomeric base pairs and provide a feasible pathway for spontaneous DNA mutations.
Collapse
Affiliation(s)
- Louie Slocombe
- grid.5475.30000 0004 0407 4824Leverhulme Quantum Biology Doctoral Training Centre, University of Surrey, Guildford, GU2 7XH UK ,grid.5475.30000 0004 0407 4824Department of Chemistry, University of Surrey, Guildford, GU2 7XH UK
| | - Max Winokan
- grid.5475.30000 0004 0407 4824Leverhulme Quantum Biology Doctoral Training Centre, University of Surrey, Guildford, GU2 7XH UK
| | - Jim Al-Khalili
- grid.5475.30000 0004 0407 4824Department of Physics, University of Surrey, Guildford, GU2 7XH UK
| | - Marco Sacchi
- grid.5475.30000 0004 0407 4824Department of Chemistry, University of Surrey, Guildford, GU2 7XH UK
| |
Collapse
|
|
16
|
Rattaprasert P, Suntornthiticharoen P, Limudomporn P, Thima K, Chavalitshewinkoon-Petmitr P. Inhibitory effects of anthracyclines on partially purified 5'-3' DNA helicase of Plasmodium falciparum. Malar J 2022; 21:216. [PMID: 35821133 PMCID: PMC9275250 DOI: 10.1186/s12936-022-04238-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2022] [Accepted: 07/04/2022] [Indexed: 11/29/2022] Open
Abstract
Background Plasmodium falciparum has been becoming resistant to the currently used anti-malarial drugs. Searching for new drug targets is urgently needed for anti-malarial development. DNA helicases separating double-stranded DNA into single-stranded DNA intermediates are essential in nearly all DNA metabolic transactions, thus they may act as a candidate for new drug targets against malarial parasites. Methods In this study, a P. falciparum 5′ to 3′ DNA helicase (PfDH-B) was partially purified from the crude extract of chloroquine- and pyrimethamine-resistant P. falciparum strain K1, by ammonium sulfate precipitation and three chromatographic procedures. DNA helicase activity of partially purified PfDH-B was examined by measuring its ability to unwind 32P-labelled partial duplex DNA. The directionality of PfDH-B was determined, and substrate preference was tested by using various substrates. Inhibitory effects of DNA intercalators such as anthracycline antibiotics on PfDH-B unwinding activity and parasite growth were investigated. Results The native PfDH-B was partially purified with a specific activity of 4150 units/mg. The PfDH-B could unwind M13-17-mer, M13-31-mer with hanging tail at 3′ or 5′ end and a linear substrate with 3′ end hanging tail but not blunt-ended duplex DNA, and did not need a fork-like substrate. Anthracyclines including aclarubicin, daunorubicin, doxorubicin, and nogalamycin inhibited the unwinding activity of PfDH-B with an IC50 value of 4.0, 7.5, 3.6, and 3.1 µM, respectively. Nogalamycin was the most effective inhibitor on PfDH-B unwinding activity and parasite growth (IC50 = 0.1 ± 0.002 µM). Conclusion Partial purification and characterization of 5′–3′ DNA helicase of P. falciparum was successfully performed. The partially purified PfDH-B does not need a fork-like substrate structure found in P. falciparum 3′ to 5′ DNA helicase (PfDH-A). Interestingly, nogalamycin was the most potent anthracycline inhibitor for PfDH-B helicase activity and parasite growth in culture. Further studies are needed to search for more potent but less cytotoxic inhibitors targeting P. falciparum DNA helicase in the future.
Collapse
Affiliation(s)
- Pongruj Rattaprasert
- Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Ratchawithi Road, Bangkok, 10400, Thailand
| | | | - Paviga Limudomporn
- Department of Zoology, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand
| | - Kanthinich Thima
- Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Ratchawithi Road, Bangkok, 10400, Thailand
| | | |
Collapse
|
|
17
|
The convergence of head-on DNA unwinding forks induces helicase oligomerization and activity transition. Proc Natl Acad Sci U S A 2022; 119:e2116462119. [PMID: 35658074 DOI: 10.1073/pnas.2116462119] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
SignificanceBloom syndrome helicase (BLM) is a multifunctional helicase that primarily catalyzes the separation of two single strands of DNA. Here, using a single-molecule optical tweezers approach combined with confocal microscopy, we monitored both the enzymatic activity and oligomeric status of BLM at the same time. Strikingly, a head-on collision of BLM-medicated DNA unwinding forks was found to effectively switch their oligomeric state and activity. Specifically, BLMs, upon collision, immediately fuse across the fork junctions and covert their activities from dsDNA unwinding to ssDNA translocation and protein displacement. These findings explain how BLM plays multiple functional roles in homologous recombination (HR). The single-molecule approach used here provides a reference model for investigating the relationship between protein oligomeric state and function.
Collapse
|
|
18
|
Lin J, Shorter J, Lucius AL. AAA+ proteins: one motor, multiple ways to work. Biochem Soc Trans 2022; 50:895-906. [PMID: 35356966 PMCID: PMC9115847 DOI: 10.1042/bst20200350] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2021] [Revised: 03/14/2022] [Accepted: 03/15/2022] [Indexed: 12/15/2022]
Abstract
Numerous ATPases associated with diverse cellular activities (AAA+) proteins form hexameric, ring-shaped complexes that function via ATPase-coupled translocation of substrates across the central channel. Cryo-electron microscopy of AAA+ proteins processing substrate has revealed non-symmetric, staircase-like hexameric structures that indicate a sequential clockwise/2-residue step translocation model for these motors. However, for many of the AAA+ proteins that share similar structural features, their translocation properties have not yet been experimentally determined. In the cases where translocation mechanisms have been determined, a two-residue translocation step-size has not been resolved. In this review, we explore Hsp104, ClpB, ClpA and ClpX as examples to review the experimental methods that have been used to examine, in solution, the translocation mechanisms employed by AAA+ motor proteins. We then ask whether AAA+ motors sharing similar structural features can have different translocation mechanisms. Finally, we discuss whether a single AAA+ motor can adopt multiple translocation mechanisms that are responsive to different challenges imposed by the substrate or the environment. We suggest that AAA+ motors adopt more than one translocation mechanism and are tuned to switch to the most energetically efficient mechanism when constraints are applied.
Collapse
Affiliation(s)
- JiaBei Lin
- Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A
| | - James Shorter
- Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A
| | - Aaron L. Lucius
- Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL, U.S.A
| |
Collapse
|
|
19
|
Chmielewska-Jeznach M, Steczkiewicz K, Kobyłecki K, Bardowski JK, Szczepankowska AK. An Adenosine Triphosphate- Dependent 5'-3' DNA Helicase From sk1-Like Lactococcus lactis F13 Phage. Front Microbiol 2022; 13:840219. [PMID: 35369496 PMCID: PMC8965321 DOI: 10.3389/fmicb.2022.840219] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2021] [Accepted: 01/31/2022] [Indexed: 11/21/2022] Open
Abstract
Here, we describe functional characterization of an early gene (gp46) product of a virulent Lactococcus lactis sk1-like phage, vB_Llc_bIBBF13 (abbr. F13). The GP46F13 protein carries a catalytically active RecA-like domain belonging to the P-loop NTPase superfamily. It also retains features characteristic for ATPases forming oligomers. In order to elucidate its detailed molecular function, we cloned and overexpressed the gp46 gene in Escherichia coli. Purified GP46F13 protein binds to DNA and exhibits DNA unwinding activity on branched substrates in the presence of adenosine triphosphate (ATP). Size exclusion chromatography with multi-angle light scattering (SEC-MALS) experiments demonstrate that GP46F13 forms oligomers, and further pull-down assays show that GP46F13 interacts with host proteins involved in replication (i.e., DnaK, DnaJ, topoisomerase I, and single-strand binding protein). Taking together the localization of the gene and the obtained results, GP46F13 is the first protein encoded in the early-expressed gene region with helicase activity that has been identified among lytic L. lactis phages up to date.
Collapse
Affiliation(s)
| | - Kamil Steczkiewicz
- Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland
| | - Kamil Kobyłecki
- Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland
| | - Jacek K Bardowski
- Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland
| | | |
Collapse
|
|
20
|
Gao B, Zheng YT, Su AM, Sun B, Xi XG, Hou XM. Remodeling the conformational dynamics of I-motif DNA by helicases in ATP-independent mode at acidic environment. iScience 2022; 25:103575. [PMID: 34988409 PMCID: PMC8704484 DOI: 10.1016/j.isci.2021.103575] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2021] [Revised: 10/09/2021] [Accepted: 12/03/2021] [Indexed: 01/18/2023] Open
Abstract
I-motifs are noncanonical four-stranded DNA structures formed by C-rich sequences at acidic environment with critical biofunctions. The particular pH sensitivity has inspired the development of i-motifs as pH sensors and DNA motors in nanotechnology. However, the folding and regulation mechanisms of i-motifs remain elusive. Here, using single-molecule FRET, we first show that i-motifs are more dynamic than G4s. Impressively, i-motifs display a high diversity of six folding species with slow interconversion. Further results indicate that i-motifs can be linearized by Replication protein A. More importantly, we identified a number of helicases with high specificity to i-motifs at low pH. All these helicases directly act on and efficiently resolve i-motifs into intermediates independent of ATP, although they poorly unwind G4 or duplex at low pH. Owing to the extreme sensitivity to helicases and no need for ATP, i-motif may be applied as a probe for helicase sensing both in vitro and in vivo.
Collapse
Affiliation(s)
- Bo Gao
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling 712100, China
| | - Ya-Ting Zheng
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling 712100, China
| | - Ai-Min Su
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling 712100, China
| | - Bo Sun
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
| | - Xu-Guang Xi
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling 712100, China
- LBPA, Ecole Normale Supérieure Paris-Saclay, CNRS, Gif-sur-Yvette, France
| | - Xi-Miao Hou
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling 712100, China
- Corresponding author
| |
Collapse
|
|
21
|
Bi L, Qin Z, Hou XM, Modesti M, Sun B. Simultaneous Mechanical and Fluorescence Detection of Helicase-Catalyzed DNA Unwinding. Methods Mol Biol 2022; 2478:329-347. [PMID: 36063326 DOI: 10.1007/978-1-0716-2229-2_12] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
Helicases are ubiquitous molecular motor proteins that utilize the energy derived from the hydrolysis of nucleoside triphosphates (NTPs) to transiently convert the duplex form of nucleic acids to single-stranded intermediates for many biological processes. These enzymes play vital roles in nearly all aspects of nucleic acid metabolism, such as DNA repair and RNA splicing. Understanding helicase's functional roles requires methods to dissect the mechanisms of motor proteins at the molecular level. In the past three decades, there has been a large increase in the application of single-molecule approaches to investigate helicases. These techniques, such as optical tweezers and single-molecule fluorescence, offer capabilities to monitor helicase motions with unprecedented spatiotemporal resolution, to apply quantitative forces to probe the chemo-mechanical activities of these motors and to resolve helicase heterogeneity at the single-molecule level. In this chapter, we describe a single-molecule method that combines optical tweezers with confocal fluorescence microscopy to study helicase-catalyzed DNA unwinding. Using Bloom syndrome protein (BLM), a multifunctional helicase that maintains genome stability, as an example, we show that this method allows for the simultaneous detection of displacement, force and fluorescence signals of a single DNA molecule during unwinding in real time, leading to the discovery of a distinct bidirectional unwinding mode of BLM that is activated by a single-stranded DNA binding protein called replication protein A (RPA). We provide detailed instructions on how to prepare two DNA templates to be used in the assays, purify the BLM and RPA proteins, perform single-molecule experiments, and acquire and analyse the data.
Collapse
Affiliation(s)
- Lulu Bi
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Zhenheng Qin
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Xi-Miao Hou
- College of Life Sciences, Northwest A&F University, Yangling, China
| | - Mauro Modesti
- Cancer Research Center of Marseille, Marseille, France
| | - Bo Sun
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
| |
Collapse
|
|
22
|
Yokota H. Quantitative and kinetic single-molecule analysis of DNA unwinding by <i>Escherichia coli</i> UvrD helicase. Biophys Physicobiol 2022; 19:1-16. [PMID: 35435650 PMCID: PMC8967476 DOI: 10.2142/biophysico.bppb-v19.0006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Accepted: 03/07/2022] [Indexed: 12/01/2022] Open
Abstract
Helicases are nucleic acid-unwinding enzymes involved in the maintenance of genome integrity. Helicases share several “helicase motifs” that are highly conserved amino acid sequences and are classified into six superfamilies (SFs). The helicase SFs are further grouped into two classes based on their functional units. One class that includes SFs 3–6 functions as a hexamer that can form a ring around DNA. Another class that includes SFs 1 and 2 functions in a non-hexameric form. The high homology in the primary and tertiary structures among SF1 helicases suggests that SF1 helicases have a common underlying mechanism. However, two opposing models for the functional unit, monomer and dimer models, have been proposed to explain DNA unwinding by SF1 helicases. This paper briefly describes the classification of helicase SFs and discusses the structural homology and the two opposing non-hexameric helicase models of SF1 helicases by focusing on Escherichia coli SF1 helicase UvrD, which plays a significant role in both nucleotide-excision repair and methyl-directed mismatch repair. This paper reviews past and recent studies on UvrD, including the author's single-molecule direct visualization of wild-type UvrD and a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C), the latter of which was used in genetic and biochemical assays that supported the monomer model. The visualization revealed that multiple UvrDΔ40C molecules jointly unwind DNA, presumably in an oligomeric form, similar to wild-type UvrD. Therefore, single-molecule direct visualization of nucleic acid-binding proteins can provide quantitative and kinetic information to reveal their fundamental mechanisms.
Collapse
Affiliation(s)
- Hiroaki Yokota
- The Graduate School for the Creation of New Photonics Industries
| |
Collapse
|
|
23
|
Mehyar N, Mashhour A, Islam I, Alhadrami HA, Tolah AM, Alghanem B, Alkhaldi S, Somaie BA, Al Ghobain M, Alobaida Y, Alaskar AS, Boudjelal M. Discovery of Zafirlukast as a novel SARS-CoV-2 helicase inhibitor using in silico modelling and a FRET-based assay. SAR AND QSAR IN ENVIRONMENTAL RESEARCH 2021; 32:963-983. [PMID: 34818959 DOI: 10.1080/1062936x.2021.1993995] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/12/2021] [Accepted: 10/12/2021] [Indexed: 06/13/2023]
Abstract
The coronavirus helicase is an essential enzyme required for viral replication/transcription pathways. Structural studies revealed a sulphate moiety that interacts with key residues within the nucleotide-binding site of the helicase. Compounds with a sulphoxide or a sulphone moiety could interfere with these interactions and consequently inhibit the enzyme. The molecular operating environment (MOE) was used to dock 189 sulphoxide and sulphone-containing FDA-approved compounds to the nucleotide-binding site. Zafirlukast, a leukotriene receptor antagonist used to treat chronic asthma, achieved the lowest docking score at -8.75 kcals/mol. The inhibitory effect of the compounds on the SARS-CoV-2 helicase dsDNA unwinding activity was tested by a FRET-based assay. Zafirlukast was the only compound to inhibit the enzyme (IC50 = 16.3 µM). The treatment of Vero E6 cells with 25 µM zafirlukast prior to SARS-CoV-2 infection decreased the cytopathic effects of SARS-CoV-2 significantly. These results suggest that zafirlukast alleviates SARS-CoV-2 pathogenicity by inhibiting the viral helicase and impairing the viral replication/transcription pathway. Zafirlukast could be clinically developed as a new antiviral treatment for SARS-CoV-2 and other coronavirus diseases. This discovery is based on molecular modelling, in vitro inhibition of the SARS-CoV helicase activity and cell-based SARS-CoV-2 viral replication.
Collapse
Affiliation(s)
- N Mehyar
- King Abdullah International Medical Research Center, Riyadh, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
- King Abdulaziz Medical City, Ministry of National Guard-Health Affairs, Riyadh, Saudi Arabia
| | - A Mashhour
- King Abdullah International Medical Research Center, Riyadh, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
- King Abdulaziz Medical City, Ministry of National Guard-Health Affairs, Riyadh, Saudi Arabia
| | - I Islam
- King Abdullah International Medical Research Center, Riyadh, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
- King Abdulaziz Medical City, Ministry of National Guard-Health Affairs, Riyadh, Saudi Arabia
| | - H A Alhadrami
- Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Rabigh, Saudi Arabia
- Molecular Diagnostic Laboratory, King Abdulaziz University Hospital, King Abdulaziz University, Jeddah, Saudi Arabia
- Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
| | - A M Tolah
- Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Rabigh, Saudi Arabia
- Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
| | - B Alghanem
- King Abdullah International Medical Research Center, Riyadh, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
- King Abdulaziz Medical City, Ministry of National Guard-Health Affairs, Riyadh, Saudi Arabia
| | - S Alkhaldi
- King Abdullah International Medical Research Center, Riyadh, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
- King Abdulaziz Medical City, Ministry of National Guard-Health Affairs, Riyadh, Saudi Arabia
| | - B A Somaie
- King Abdullah International Medical Research Center, Riyadh, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
- King Abdulaziz Medical City, Ministry of National Guard-Health Affairs, Riyadh, Saudi Arabia
| | - M Al Ghobain
- King Abdullah International Medical Research Center, Riyadh, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
- King Abdulaziz Medical City, Ministry of National Guard-Health Affairs, Riyadh, Saudi Arabia
| | - Y Alobaida
- Sudair Pharmaceutical Co, Riyadh, Saudi Arabia
| | - A S Alaskar
- King Abdullah International Medical Research Center, Riyadh, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
- King Abdulaziz Medical City, Ministry of National Guard-Health Affairs, Riyadh, Saudi Arabia
| | - M Boudjelal
- King Abdullah International Medical Research Center, Riyadh, Saudi Arabia
- King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
- King Abdulaziz Medical City, Ministry of National Guard-Health Affairs, Riyadh, Saudi Arabia
| |
Collapse
|
|
24
|
Abstract
Ring-shaped hexameric helicases are essential motor proteins that separate duplex nucleic acid strands for DNA replication, recombination, and transcriptional regulation. Two evolutionarily distinct lineages of these enzymes, predicated on RecA and AAA+ ATPase folds, have been identified and characterized to date. Hexameric helicases couple NTP hydrolysis with conformational changes that move nucleic acid substrates through a central pore in the enzyme. How hexameric helicases productively engage client DNA or RNA segments and use successive rounds of NTPase activity to power translocation and unwinding have been longstanding questions in the field. Recent structural and biophysical findings are beginning to reveal commonalities in NTP hydrolysis and substrate translocation by diverse hexameric helicase families. Here, we review these molecular mechanisms and highlight aspects of their function that are yet to be understood.
Collapse
|
|
25
|
Liu Y, Zhu X, Wang K, Zhang B, Qiu S. The Cellular Functions and Molecular Mechanisms of G-Quadruplex Unwinding Helicases in Humans. Front Mol Biosci 2021; 8:783889. [PMID: 34912850 PMCID: PMC8667583 DOI: 10.3389/fmolb.2021.783889] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2021] [Accepted: 11/02/2021] [Indexed: 01/19/2023] Open
Abstract
G-quadruplexes (G4s) are stable non-canonical secondary structures formed by G-rich DNA or RNA sequences. They play various regulatory roles in many biological processes. It is commonly agreed that G4 unwinding helicases play key roles in G4 metabolism and function, and these processes are closely related to physiological and pathological processes. In recent years, more and more functional and mechanistic details of G4 helicases have been discovered; therefore, it is necessary to carefully sort out the current research efforts. Here, we provide a systematic summary of G4 unwinding helicases from the perspective of functions and molecular mechanisms. First, we provide a general introduction about helicases and G4s. Next, we comprehensively summarize G4 unfolding helicases in humans and their proposed cellular functions. Then, we review their study methods and molecular mechanisms. Finally, we share our perspective on further prospects. We believe this review will provide opportunities for researchers to reach the frontiers in the functions and molecular mechanisms of human G4 unwinding helicases.
Collapse
Affiliation(s)
- Yang Liu
- Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Collaborative Innovation Center for Mountain Ecology and Agro-Bioengineering (CICMEAB), College of Life Sciences/Institute of Agro-bioengineering, Guizhou University, Guiyang, China
- College of Basic Medicine, Zunyi Medical University, Zunyi, China
- The Key Laboratory of Fermentation Engineering and Biological Pharmacy of Guizhou Province, Guizhou University, Guiyang, China
- School of Liquor and Food Engineering, Guizhou University, Guiyang, China
| | - Xinting Zhu
- College of Basic Medicine, Zunyi Medical University, Zunyi, China
| | - Kejia Wang
- Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Collaborative Innovation Center for Mountain Ecology and Agro-Bioengineering (CICMEAB), College of Life Sciences/Institute of Agro-bioengineering, Guizhou University, Guiyang, China
- The Key Laboratory of Fermentation Engineering and Biological Pharmacy of Guizhou Province, Guizhou University, Guiyang, China
- School of Liquor and Food Engineering, Guizhou University, Guiyang, China
| | - Bo Zhang
- College of Basic Medicine, Zunyi Medical University, Zunyi, China
| | - Shuyi Qiu
- Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Collaborative Innovation Center for Mountain Ecology and Agro-Bioengineering (CICMEAB), College of Life Sciences/Institute of Agro-bioengineering, Guizhou University, Guiyang, China
- The Key Laboratory of Fermentation Engineering and Biological Pharmacy of Guizhou Province, Guizhou University, Guiyang, China
- School of Liquor and Food Engineering, Guizhou University, Guiyang, China
| |
Collapse
|
|
26
|
Determining translocation orientations of nucleic acid helicases. Methods 2021; 204:160-171. [PMID: 34758393 PMCID: PMC9076756 DOI: 10.1016/j.ymeth.2021.11.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2021] [Revised: 10/29/2021] [Accepted: 11/02/2021] [Indexed: 11/20/2022] Open
Abstract
Helicase enzymes translocate along an RNA or DNA template with a defined polarity to unwind, separate, or remodel duplex strands for a variety of genome maintenance processes. Helicase mutations are commonly associated with a variety of diseases including aging, cancer, and neurodegeneration. Biochemical characterization of these enzymes has provided a wealth of information on the kinetics of unwinding and substrate preferences, and several high-resolution structures of helicases alone and bound to oligonucleotides have been solved. Together, they provide mechanistic insights into the structural translocation and unwinding orientations of helicases. However, these insights rely on structural inferences derived from static snapshots. Instead, continued efforts should be made to combine structure and kinetics to better define active translocation orientations of helicases. This review explores many of the biochemical and biophysical methods utilized to map helicase binding orientation to DNA or RNA substrates and includes several time-dependent methods to unequivocally map the active translocation orientation of these enzymes to better define the active leading and trailing faces.
Collapse
|
|
27
|
Yang XW, Liu J. Observing Protein One-Dimensional Sliding: Methodology and Biological Significance. Biomolecules 2021; 11:1618. [PMID: 34827616 PMCID: PMC8615959 DOI: 10.3390/biom11111618] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2021] [Revised: 10/15/2021] [Accepted: 10/16/2021] [Indexed: 11/28/2022] Open
Abstract
One-dimensional (1D) sliding of DNA-binding proteins has been observed by numerous kinetic studies. It appears that many of these sliding events play important roles in a wide range of biological processes. However, one challenge is to determine the physiological relevance of these motions in the context of the protein's biological function. Here, we discuss methods of measuring protein 1D sliding by highlighting the single-molecule approaches that are capable of visualizing particle movement in real time. We also present recent findings that show how protein sliding contributes to function.
Collapse
Affiliation(s)
| | - Jiaquan Liu
- State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China;
| |
Collapse
|
|
28
|
Shi R, Hou W, Wang ZQ, Xu X. Biogenesis of Iron-Sulfur Clusters and Their Role in DNA Metabolism. Front Cell Dev Biol 2021; 9:735678. [PMID: 34660592 PMCID: PMC8514734 DOI: 10.3389/fcell.2021.735678] [Citation(s) in RCA: 46] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2021] [Accepted: 09/06/2021] [Indexed: 12/02/2022] Open
Abstract
Iron–sulfur (Fe/S) clusters (ISCs) are redox-active protein cofactors that their synthesis, transfer, and insertion into target proteins require many components. Mitochondrial ISC assembly is the foundation of all cellular ISCs in eukaryotic cells. The mitochondrial ISC cooperates with the cytosolic Fe/S protein assembly (CIA) systems to accomplish the cytosolic and nuclear Fe/S clusters maturation. ISCs are needed for diverse cellular functions, including nitrogen fixation, oxidative phosphorylation, mitochondrial respiratory pathways, and ribosome assembly. Recent research advances have confirmed the existence of different ISCs in enzymes that regulate DNA metabolism, including helicases, nucleases, primases, DNA polymerases, and glycosylases. Here we outline the synthesis of mitochondrial, cytosolic and nuclear ISCs and highlight their functions in DNA metabolism.
Collapse
Affiliation(s)
- Ruifeng Shi
- Shenzhen University-Friedrich Schiller Universität Jena Joint Ph.D. Program in Biomedical Sciences, Shenzhen University School of Medicine, Shenzhen, China.,Guangdong Key Laboratory for Genome Stability and Disease Prevention and Marshall Laboratory of Biomedical Engineering, Shenzhen University School of Medicine, Shenzhen, China
| | - Wenya Hou
- Guangdong Key Laboratory for Genome Stability and Disease Prevention and Marshall Laboratory of Biomedical Engineering, Shenzhen University School of Medicine, Shenzhen, China
| | - Zhao-Qi Wang
- Leibniz Institute on Aging-Fritz Lipmann Institute (FLI), Jena, Germany.,Faculty of Biological Sciences, Friedrich-Schiller-University Jena, Jena, Germany
| | - Xingzhi Xu
- Shenzhen University-Friedrich Schiller Universität Jena Joint Ph.D. Program in Biomedical Sciences, Shenzhen University School of Medicine, Shenzhen, China.,Guangdong Key Laboratory for Genome Stability and Disease Prevention and Marshall Laboratory of Biomedical Engineering, Shenzhen University School of Medicine, Shenzhen, China
| |
Collapse
|
|
29
|
Single-molecule studies of helicases and translocases in prokaryotic genome-maintenance pathways. DNA Repair (Amst) 2021; 108:103229. [PMID: 34601381 DOI: 10.1016/j.dnarep.2021.103229] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2021] [Revised: 09/13/2021] [Accepted: 09/16/2021] [Indexed: 11/22/2022]
Abstract
Helicases involved in genomic maintenance are a class of nucleic-acid dependent ATPases that convert the energy of ATP hydrolysis into physical work to execute irreversible steps in DNA replication, repair, and recombination. Prokaryotic helicases provide simple models to understand broadly conserved molecular mechanisms involved in manipulating nucleic acids during genome maintenance. Our understanding of the catalytic properties, mechanisms of regulation, and roles of prokaryotic helicases in DNA metabolism has been assembled through a combination of genetic, biochemical, and structural methods, further refined by single-molecule approaches. Together, these investigations have constructed a framework for understanding the mechanisms that maintain genomic integrity in cells. This review discusses recent single-molecule insights into molecular mechanisms of prokaryotic helicases and translocases.
Collapse
|
|
30
|
Gnanasekaran P, Gupta N, Ponnusamy K, Chakraborty S. Geminivirus Betasatellite-Encoded βC1 Protein Exhibits Novel ATP Hydrolysis Activity That Influences Its DNA-Binding Activity and Viral Pathogenesis. J Virol 2021; 95:e0047521. [PMID: 34132576 PMCID: PMC8354231 DOI: 10.1128/jvi.00475-21] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Accepted: 06/09/2021] [Indexed: 11/20/2022] Open
Abstract
Plant virus satellites are maintained by their associated helper viruses, and satellites influence viral pathogenesis. Diseases caused by geminivirus-betasatellite complexes can become epidemics and therefore have become a threat to economically important crops across the world. Here, we identified a novel molecular function of the betasatellite-encoded pathogenicity determinant βC1. The tomato leaf curl Patna betasatellite (ToLCPaB)-encoded βC1 protein was found to exhibit novel ATPase activity in the presence of the divalent metal ion cofactor MgCl2. Moreover, ATPase activity was confirmed to be ubiquitously displayed by βC1 proteins encoded by diverse betasatellites. Mutational and sequence analysis showed that conserved lysine/arginine residues at positions 49/50 and 91 of βC1 proteins are essential for their ATPase activity. Biochemical studies revealed that the DNA-binding activity of the βC1 protein was interfered with by the binding of ATP to the protein. Mutating arginine 91 of βC1 to alanine reduced its DNA-binding activity. The results of docking studies provided evidence for an overlap of the ATP-binding and DNA-binding regions of βC1 and for the importance of arginine 91 for both ATP-binding and DNA-binding activities. A mutant betasatellite with a specifically βC1-ATPase dominant negative mutation was found to induce symptoms on Nicotiana benthamiana plants similar to those induced by wild-type betasatellite infection. The ATPase function of βC1 was found to be negatively associated with geminivirus-betasatellite DNA accumulation, despite the positive influence of this ATPase function on the accumulation of replication-associated protein (Rep) and βC1 transcripts. IMPORTANCE Most satellites influence the pathogenesis of their helper viruses. Here, we characterized the novel molecular function of βC1, a nonstructural pathogenicity determinant protein encoded by a betasatellite. We demonstrated the display of ATPase activity by this βC1 protein. Additionally, we confirmed the ubiquitous display of ATPase activity by βC1 proteins encoded by diverse betasatellites. The lysine/arginine residues conserved at positions 49 and 91 of βC1 were found to be crucial for its ATPase function. DNA-binding activity of βC1 was found to be reduced in the presence of ATP. Inhibition of ATPase activity of βC1 in the presence of an excess concentration of cold ATP, GTP, CTP, or UTP suggested that the purified βC1 can also hydrolyze other cellular nucleoside triphosphates (NTPs) besides ATP in vitro. These results established the importance of the ATPase and DNA-binding activities of the βC1 protein in regulating geminivirus-betasatellite DNA accumulation in the infected plant cell.
Collapse
Affiliation(s)
- Prabu Gnanasekaran
- Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru Universitygrid.10706.30, New Delhi, India
| | - Neha Gupta
- Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru Universitygrid.10706.30, New Delhi, India
| | | | - Supriya Chakraborty
- Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru Universitygrid.10706.30, New Delhi, India
| |
Collapse
|
|
31
|
Nguyen B, Shinn MK, Weiland E, Lohman TM. Regulation of E. coli Rep helicase activity by PriC. J Mol Biol 2021; 433:167072. [PMID: 34081984 PMCID: PMC8941637 DOI: 10.1016/j.jmb.2021.167072] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2021] [Revised: 05/11/2021] [Accepted: 05/20/2021] [Indexed: 11/28/2022]
Abstract
Stalled DNA replication forks can result in incompletely replicated genomes and cell death. DNA replication restart pathways have evolved to deal with repair of stalled forks and E. coli Rep helicase functions in this capacity. Rep and an accessory protein, PriC, assemble at a stalled replication fork to facilitate loading of other replication proteins. A Rep monomer is a rapid and processive single stranded (ss) DNA translocase but needs to be activated to function as a helicase. Activation of Rep in vitro requires self-assembly to form a dimer, removal of its auto-inhibitory 2B sub-domain, or interactions with an accessory protein. Rep helicase activity has been shown to be stimulated by PriC, although the mechanism of activation is not clear. Using stopped flow kinetics, analytical sedimentation and single molecule fluorescence methods, we show that a PriC dimer activates the Rep monomer helicase and can also stimulate the Rep dimer helicase. We show that PriC can self-assemble to form dimers and tetramers and that Rep and PriC interact in the absence of DNA. We further show that PriC serves as a Rep processivity factor, presumably co-translocating with Rep during DNA unwinding. Activation is specific for Rep since PriC does not activate the UvrD helicase. Interaction of PriC with the C-terminal acidic tip of the ssDNA binding protein, SSB, eliminates Rep activation by stabilizing the PriC monomer. This suggests a likely mechanism for Rep activation by PriC at a stalled replication fork.
Collapse
Affiliation(s)
- Binh Nguyen
- Department of Biochemistry and Molecular Biophysics, Box 8231, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, United States
| | - Min Kyung Shinn
- Department of Physics, Washington University in St. Louis, St. Louis, MO 63130, United States
| | - Elizabeth Weiland
- Department of Biochemistry and Molecular Biophysics, Box 8231, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, United States
| | - Timothy M Lohman
- Department of Biochemistry and Molecular Biophysics, Box 8231, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, United States.
| |
Collapse
|
|
32
|
Dai YX, Chen WF, Liu NN, Teng FY, Guo HL, Hou XM, Dou SX, Rety S, Xi XG. Structural and functional studies of SF1B Pif1 from Thermus oshimai reveal dimerization-induced helicase inhibition. Nucleic Acids Res 2021; 49:4129-4143. [PMID: 33784404 PMCID: PMC8053095 DOI: 10.1093/nar/gkab188] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Revised: 03/03/2021] [Accepted: 03/05/2021] [Indexed: 01/06/2023] Open
Abstract
Pif1 is an SF1B helicase that is evolutionarily conserved from bacteria to humans and plays multiple roles in maintaining genome stability in both nucleus and mitochondria. Though highly conserved, Pif1 family harbors a large mechanistic diversity. Here, we report crystal structures of Thermus oshimai Pif1 (ToPif1) alone and complexed with partial duplex or single-stranded DNA. In the apo state and in complex with a partial duplex DNA, ToPif1 is monomeric with its domain 2B/loop3 adopting a closed and an open conformation, respectively. When complexed with a single-stranded DNA, ToPif1 forms a stable dimer with domain 2B/loop3 shifting to a more open conformation. Single-molecule and biochemical assays show that domain 2B/loop3 switches repetitively between the closed and open conformations when a ToPif1 monomer unwinds DNA and, in contrast with other typical dimeric SF1A helicases, dimerization has an inhibitory effect on its helicase activity. This mechanism is not general for all Pif1 helicases but illustrates the diversity of regulation mechanisms among different helicases. It also raises the possibility that although dimerization results in activation for SF1A helicases, it may lead to inhibition for some of the other uncharacterized SF1B helicases, an interesting subject warranting further studies.
Collapse
Affiliation(s)
- Yang-Xue Dai
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Wei-Fei Chen
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Na-Nv Liu
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Fang-Yuan Teng
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Hai-Lei Guo
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Xi-Miao Hou
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Shuo-Xing Dou
- Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.,School of Physical Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Stephane Rety
- Univ. Lyon, ENS de Lyon, Univ. Claude Bernard CNRS UMR 5239, INSERM U1210, LBMC, 46 allée d'Italie Site Jacques Monod, F-69007, Lyon, France
| | - Xu-Guang Xi
- State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China.,Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), UMR 8113 CNRS, Institut D'Alembert, École Normale Supérieure Paris-Saclay, Université Paris-Saclay, 4, Avenue des Sciences, 91190 Gif sur Yvette, France
| |
Collapse
|
|
33
|
Zhu XH, Sun BF, Luo M, Yu J, Zhang YD, Xu HQ, Luo H. Bloom helicase explicitly unwinds 3'-tailed G4DNA structure in prostate cancer cells. Int J Biol Macromol 2021; 180:578-589. [PMID: 33727188 DOI: 10.1016/j.ijbiomac.2021.03.060] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2020] [Revised: 01/22/2021] [Accepted: 03/11/2021] [Indexed: 10/21/2022]
Abstract
G-quadruplex DNA (G4DNA) structure, which widely exists in the chromosomal telomeric regions and oncogenic promoter regions, plays a pivotal role in extending telomeric DNA with the help of telomerase in human cells. Bloom (BLM) helicase, a crucial member of the family of genome surveillance proteins, plays an essential role in DNA metabolic and repair pathways, including DNA replication, repair, transcription, recombination during chromosome segregation, and assuring telomere stability. The unwinding of G4DNA requires the participation of DNA helicase, which is crucial for maintaining chromosomal stability in cancer cells. Using fluorescence polarization and the electrophoretic mobility shift assay (EMSA), this study aimed to investigate the DNA-binding and unwinding properties of BLM helicase, cloned and purified from prostate cancer cells, toward G4DNA. The results revealed that BLM helicase derived from prostate cancer cells could bind and unwind G4DNA. The molecular affinity of bond between G4DNA and the helicase was dependent on the single-stranded DNA (ssDNA) terminals in G4DNA; the helicase was effectively bound to the G4DNA when the helicase monomer sufficiently covered approximately 10 nucleotides at the 3' or 5' ssDNA tail of G4DNA. For the unwinding of G4DNA, there was an apparent requirement of a 3' ssDNA tail and ATP; a G4DNA with only a 3' ssDNA tail was identified to be the most suitable substrate to be unwound by BLM helicase and required 3' ssDNA tails of at least 10 nt in length for efficient unwinding. Besides, BLM helicase was loosely bound and partly unwound the blunt-ended G4DNA. Although further mechanistic studies are warranted, the experimental results presented in this study are beneficial to further our understanding of the functional implication of BLM helicase in prostate cancer cells.
Collapse
Affiliation(s)
- Xu-Hui Zhu
- State Key Laboratory of Functions And Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, PR China; Beijing ChaoYang Hospital, Capital Medical University, Beijing 100016, PR China
| | - Bao-Fei Sun
- State Key Laboratory of Functions And Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, PR China
| | - Mei Luo
- State Key Laboratory of Functions And Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, PR China; The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Science, Guiyang 550014, PR China
| | - Jia Yu
- State Key Laboratory of Functions And Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, PR China; The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Science, Guiyang 550014, PR China
| | | | - Hou-Qiang Xu
- Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, College of Animal Science, Guizhou University, Guiyang 550025, PR China.
| | - Heng Luo
- State Key Laboratory of Functions And Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, PR China; The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Science, Guiyang 550014, PR China; Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, College of Animal Science, Guizhou University, Guiyang 550025, PR China.
| |
Collapse
|
|
34
|
Yokota H. Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD. Int J Mol Sci 2021; 22:ijms22031018. [PMID: 33498436 PMCID: PMC7864180 DOI: 10.3390/ijms22031018] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2020] [Revised: 01/15/2021] [Accepted: 01/18/2021] [Indexed: 12/19/2022] Open
Abstract
Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.
Collapse
Affiliation(s)
- Hiroaki Yokota
- Biophotonics Laboratory, The Graduate School for the Creation of New Photonics Industries, 1955-1, Kurematsu-cho, Nishi-ku, Hamamatsu, Shizuoka 431-1202, Japan
| |
Collapse
|
|
35
|
Li D, Xu HQ, Xi XG. Macromolecular aging: ATP hydrolysis-driven functional and structural changes in Escherichia coli RecQ helicase. Biochem Biophys Res Commun 2021; 542:29-33. [PMID: 33485211 DOI: 10.1016/j.bbrc.2021.01.020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2020] [Accepted: 01/06/2021] [Indexed: 11/21/2022]
Abstract
Aging has been considered a phenomenon that can be only applied to cells or organisms. Here, we show that RecQ helicase from E. coli displays an aging phenomenon: this macromolecular motor loses its structure and function after hydrolyzing a certain number of ATP molecules. The aging process was only triggered by repeated catalytic cycles. These observations lead to a new concept: macromolecule aging.
Collapse
Affiliation(s)
- Dan Li
- College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, 712100, China
| | - Hou-Qiang Xu
- Key Laboratory of Animal Genetics Breeding and Production in the Plateau Mountains Region, Ministry of Education, Guizhou University, Guiyang, 550025, China
| | - Xu-Guang Xi
- College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, 712100, China; The LBPA, Ecole Normale Supérieure Paris-Saclay, CNRS, Université Paris Saclay, Gif-sur-Yvette, France.
| |
Collapse
|
|
36
|
Chen J, Ding T, Liang W, Li T, Wu S, Xie W, Ding N, Tang Y, Ding Z. Generation and Characterization of Monoclonal Antibody Against Porcine Epidemic Diarrhea Virus Nonstructural Protein 13. Monoclon Antib Immunodiagn Immunother 2021; 39:222-227. [PMID: 33351712 DOI: 10.1089/mab.2020.0028] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Porcine epidemic diarrhea virus (PEDV) is an enteric swine coronavirus. Recent PEDV eruption in East Asian and North American countries made it notorious and caused huge economic losses to the porcine industry. Nonstructural protein 13 (nsp13) is a nucleic acid helicase/NTPase that plays a critical role in viral gene transcription and viral replication. To investigate the function of nsp13 in the context of PEDV infection, here, PEDV nsp13 gene was amplified and cloned into pET28a/pET30a/pGEX-6P-1 expression vectors. The recombinant his-tagged nsp13 and GST-tagged nsp13 were expressed. Purified his-tagged nsp13 from pET28a-nsp13 vectors was chosen for immunization. Three monoclonal antibodies (mAbs) named 5A9, 5C7, and 5G7 were identified from the hybridoma cells, and were characterized by Western blot analysis and immunofluorescent assay, which demonstrated high specificity of these three mAbs with pCAGGS-HA-nsp13. All three mAbs belong to IgG1+ kappa subclass. However, only mAb 5A9 could effectively and specifically recognize PEDV expressing nsp13. Furthermore, the generated antibody against nsp13 could be applied to investigate nsp13 function during PEDV replication.
Collapse
Affiliation(s)
- Jun Chen
- Department of Veterinary Preventive Medicine, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, China.,Jiangxi Provincial Key Laboratory for Animal Health, Jiangxi Agricultural University, Nanchang, China
| | - Tong Ding
- Department of Veterinary Preventive Medicine, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, China.,Jiangxi Provincial Key Laboratory for Animal Health, Jiangxi Agricultural University, Nanchang, China
| | - Wenqi Liang
- Department of Veterinary Preventive Medicine, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, China.,Jiangxi Provincial Key Laboratory for Animal Health, Jiangxi Agricultural University, Nanchang, China
| | - Ting Li
- Department of Veterinary Preventive Medicine, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, China.,Jiangxi Provincial Key Laboratory for Animal Health, Jiangxi Agricultural University, Nanchang, China
| | - Shengqiang Wu
- Department of Veterinary Preventive Medicine, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, China.,Jiangxi Provincial Key Laboratory for Animal Health, Jiangxi Agricultural University, Nanchang, China
| | - Wenqing Xie
- Department of Veterinary Preventive Medicine, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, China.,Jiangxi Provincial Key Laboratory for Animal Health, Jiangxi Agricultural University, Nanchang, China
| | - Nengshui Ding
- State Key Laboratory for Pig Genetic Improvement and Production Technology, Jiangxi Agricultural University, Nanchang, China
| | - Yuxin Tang
- Department of Veterinary Preventive Medicine, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, China.,Jiangxi Provincial Key Laboratory for Animal Health, Jiangxi Agricultural University, Nanchang, China
| | - Zhen Ding
- Department of Veterinary Preventive Medicine, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, China.,Jiangxi Provincial Key Laboratory for Animal Health, Jiangxi Agricultural University, Nanchang, China
| |
Collapse
|
|
37
|
Yadav M, Singh RS, Hogan D, Vidhyasagar V, Yang S, Chung IYW, Kusalik A, Dmitriev OY, Cygler M, Wu Y. The KH domain facilitates the substrate specificity and unwinding processivity of DDX43 helicase. J Biol Chem 2021; 296:100085. [PMID: 33199368 PMCID: PMC7949032 DOI: 10.1074/jbc.ra120.015824] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2020] [Revised: 11/03/2020] [Accepted: 11/16/2020] [Indexed: 01/21/2023] Open
Abstract
The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins. Recently, we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding ssDNA and ssRNA. Electrophoretic mobility shift assay and NMR revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. Systematic evolution of ligands by exponential enrichment, chromatin immunoprecipitation-seq, and cross-linking immunoprecipitation-seq showed that the KH domain binds C-/T-rich DNA and U-rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-heteronuclear single quantum coherence NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single-stranded tails and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.
Collapse
Affiliation(s)
- Manisha Yadav
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | - Ravi Shankar Singh
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | - Daniel Hogan
- Department of Computer Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | | | - Shizhuo Yang
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | - Ivy Yeuk Wah Chung
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | - Anthony Kusalik
- Department of Computer Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | - Oleg Y Dmitriev
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | - Miroslaw Cygler
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | - Yuliang Wu
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
| |
Collapse
|
|
38
|
Mehyar N, Mashhour A, Islam I, Gul S, Adedeji AO, Askar AS, Boudjelal M. Using in silico modelling and FRET-based assays in the discovery of novel FDA-approved drugs as inhibitors of MERS-CoV helicase. SAR AND QSAR IN ENVIRONMENTAL RESEARCH 2021; 32:51-70. [PMID: 33401979 DOI: 10.1080/1062936x.2020.1857437] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/14/2020] [Accepted: 11/25/2020] [Indexed: 06/12/2023]
Abstract
A Förster resonance energy transfer (FRET)-based assay was used to screen the FDA-approved compound library against the MERS-CoV helicase, an essential enzyme for virus replication within the host cell. Five compounds inhibited the helicase activity with submicromolar potencies (IC50, 0.73-1.65 µM) and ten compounds inhibited the enzyme with micromolar potencies (IC50, 19.6-502 µM). The molecular operating environment (MOE) was used to dock the identified inhibitors on the MERS-CoV helicase nucleotide binding. Strong inhibitors docked well in the nucleotide-binding site and established interactions with some of the essential residues. There was a reasonable correlation between the observed IC50 values and the MOE docking scores of the strong inhibitors (r 2 = 0.74), indicating the ability of the in silico docking model to predict the binding of strong inhibitors. In silico docking could be a useful complementary tool used with the FRET-based assay to predict new MERS-CoV helicase inhibitors. The identified inhibitors could potentially be used in the clinical development of new antiviral treatment for MERS-CoV and other coronavirus related diseases, including coronavirus disease 2019 (COVID-19).
Collapse
Affiliation(s)
- N Mehyar
- King Abdullah International Medical Research Centre, King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Ministry of National Guard Health Affairs , Riyadh, Saudi Arabia
| | - A Mashhour
- King Abdullah International Medical Research Centre, King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Ministry of National Guard Health Affairs , Riyadh, Saudi Arabia
| | - I Islam
- King Abdullah International Medical Research Centre, King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Ministry of National Guard Health Affairs , Riyadh, Saudi Arabia
| | - S Gul
- Fraunhofer Institute for Molecular Biology and Applied Ecology IME - ScreeningPort , Hamburg, Germany
| | - A O Adedeji
- Department of Pathology and Population Medicine, College of Veterinary Medicine, Midwestern University , Glendale, Arizona, USA
| | - A S Askar
- King Abdullah International Medical Research Centre, King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Ministry of National Guard Health Affairs , Riyadh, Saudi Arabia
| | - M Boudjelal
- King Abdullah International Medical Research Centre, King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Ministry of National Guard Health Affairs , Riyadh, Saudi Arabia
| |
Collapse
|
|
39
|
Desai PR, Brahmachari S, Marko JF, Das S, Neuman KC. Coarse-grained modelling of DNA plectoneme pinning in the presence of base-pair mismatches. Nucleic Acids Res 2020; 48:10713-10725. [PMID: 33045724 DOI: 10.1093/nar/gkaa836] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2020] [Revised: 09/14/2020] [Accepted: 09/18/2020] [Indexed: 12/27/2022] Open
Abstract
Damaged or mismatched DNA bases result in the formation of physical defects in double-stranded DNA. In vivo, defects in DNA must be rapidly and efficiently repaired to maintain cellular function and integrity. Defects can also alter the mechanical response of DNA to bending and twisting constraints, both of which are important in defining the mechanics of DNA supercoiling. Here, we use coarse-grained molecular dynamics (MD) simulation and supporting statistical-mechanical theory to study the effect of mismatched base pairs on DNA supercoiling. Our simulations show that plectoneme pinning at the mismatch site is deterministic under conditions of relatively high force (>2 pN) and high salt concentration (>0.5 M NaCl). Under physiologically relevant conditions of lower force (0.3 pN) and lower salt concentration (0.2 M NaCl), we find that plectoneme pinning becomes probabilistic and the pinning probability increases with the mismatch size. These findings are in line with experimental observations. The simulation framework, validated with experimental results and supported by the theoretical predictions, provides a way to study the effect of defects on DNA supercoiling and the dynamics of supercoiling in molecular detail.
Collapse
Affiliation(s)
- Parth Rakesh Desai
- Department of Mechanical Engineering, University of Maryland, College Park, MD 20742, USA.,Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | | | - John F Marko
- Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208, USA.,Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA
| | - Siddhartha Das
- Department of Mechanical Engineering, University of Maryland, College Park, MD 20742, USA
| | - Keir C Neuman
- Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
| |
Collapse
|
|
40
|
Mutations in conserved functional domains of human RecQ helicases are associated with diseases and cancer: A review. Biophys Chem 2020; 265:106433. [DOI: 10.1016/j.bpc.2020.106433] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2020] [Revised: 07/06/2020] [Accepted: 07/11/2020] [Indexed: 12/12/2022]
|
|
41
|
Wilkinson OJ, Carrasco C, Aicart-Ramos C, Moreno-Herrero F, Dillingham MS. Bulk and single-molecule analysis of a bacterial DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor. Nucleic Acids Res 2020; 48:7991-8005. [PMID: 32621607 PMCID: PMC7430649 DOI: 10.1093/nar/gkaa562] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2020] [Revised: 06/12/2020] [Accepted: 06/19/2020] [Indexed: 11/14/2022] Open
Abstract
DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.
Collapse
Affiliation(s)
- Oliver J Wilkinson
- School of Biochemistry, Biomedical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, UK
| | - Carolina Carrasco
- Department of Macromolecular Structures, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Cantoblanco, Madrid, Spain
| | - Clara Aicart-Ramos
- Department of Macromolecular Structures, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Cantoblanco, Madrid, Spain
| | - Fernando Moreno-Herrero
- Department of Macromolecular Structures, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Cantoblanco, Madrid, Spain
| | - Mark S Dillingham
- School of Biochemistry, Biomedical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, UK
| |
Collapse
|
|
42
|
Brosh RM, Matson SW. History of DNA Helicases. Genes (Basel) 2020; 11:genes11030255. [PMID: 32120966 PMCID: PMC7140857 DOI: 10.3390/genes11030255] [Citation(s) in RCA: 61] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Revised: 02/18/2020] [Accepted: 02/20/2020] [Indexed: 12/13/2022] Open
Abstract
Since the discovery of the DNA double helix, there has been a fascination in understanding the molecular mechanisms and cellular processes that account for: (i) the transmission of genetic information from one generation to the next and (ii) the remarkable stability of the genome. Nucleic acid biologists have endeavored to unravel the mysteries of DNA not only to understand the processes of DNA replication, repair, recombination, and transcription but to also characterize the underlying basis of genetic diseases characterized by chromosomal instability. Perhaps unexpectedly at first, DNA helicases have arisen as a key class of enzymes to study in this latter capacity. From the first discovery of ATP-dependent DNA unwinding enzymes in the mid 1970's to the burgeoning of helicase-dependent pathways found to be prevalent in all kingdoms of life, the story of scientific discovery in helicase research is rich and informative. Over four decades after their discovery, we take this opportunity to provide a history of DNA helicases. No doubt, many chapters are left to be written. Nonetheless, at this juncture we are privileged to share our perspective on the DNA helicase field - where it has been, its current state, and where it is headed.
Collapse
Affiliation(s)
- Robert M. Brosh
- Section on DNA Helicases, Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA
- Correspondence: (R.M.B.J.); (S.W.M.); Tel.: +1-410-558-8578 (R.M.B.J.); +1-919-962-0005 (S.W.M.)
| | - Steven W. Matson
- Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
- Correspondence: (R.M.B.J.); (S.W.M.); Tel.: +1-410-558-8578 (R.M.B.J.); +1-919-962-0005 (S.W.M.)
| |
Collapse
|
|
43
|
Yokota H. DNA-Unwinding Dynamics of Escherichia coli UvrD Lacking the C-Terminal 40 Amino Acids. Biophys J 2020; 118:1634-1648. [PMID: 32142643 DOI: 10.1016/j.bpj.2020.02.014] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2019] [Revised: 02/06/2020] [Accepted: 02/11/2020] [Indexed: 01/18/2023] Open
Abstract
The E. coli UvrD protein is a nonhexameric DNA helicase that belongs to superfamily I and plays a crucial role in both nucleotide excision repair and methyl-directed mismatch repair. Previous data suggested that wild-type UvrD has optimal activity in its oligomeric form. However, crystal structures of the UvrD-DNA complex were only resolved for monomeric UvrD, using a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C). However, biochemical findings performed using UvrDΔ40C indicated that this mutant failed to dimerize, although its DNA-unwinding activity was comparable to that of wild-type UvrD. Although the C-terminus plays essential roles in nucleic acid binding for many proteins with helicase and dimerization activities, the exact function of the C-terminus is poorly understood. Thus, to understand the function of the C-terminal amino acids of UvrD, we performed single-molecule direct visualization. Photobleaching of dye-labeled UvrDΔ40C molecules revealed that two or three UvrDΔ40C molecules could bind simultaneously to an 18-bp double-stranded DNA with a 20-nucleotide, 3' single-stranded DNA tail in the absence of ATP. Simultaneous visualization of association/dissociation of the mutant with/from DNA and the DNA-unwinding dynamics of the mutant in the presence of ATP demonstrated that, as with wild-type UvrD, two or three UvrDΔ40C molecules were primarily responsible for DNA unwinding. The determined association/dissociation rate constants for the second bound monomer were ∼2.5-fold larger than that of wild-type UvrD. The involvement of multiple UvrDΔ40C molecules in DNA unwinding was also observed under a physiological salt concentration (200 mM NaCl). These results suggest that multiple UvrDΔ40C molecules, which may form an oligomer, play an active role in DNA unwinding in vivo and that deleting the C-terminal 40 residues altered the interaction of the second UvrD monomer with DNA without affecting the interaction with the first bound UvrD monomer.
Collapse
Affiliation(s)
- Hiroaki Yokota
- Biophotonics Laboratory, Graduate School for the Creation of New Photonics Industries, Hamamatsu, Shizuoka, Japan.
| |
Collapse
|
|
44
|
Yuan WF, Wan LY, Peng H, Zhong YM, Cai WL, Zhang YQ, Ai WB, Wu JF. The influencing factors and functions of DNA G-quadruplexes. Cell Biochem Funct 2020; 38:524-532. [PMID: 32056246 PMCID: PMC7383576 DOI: 10.1002/cbf.3505] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2019] [Revised: 01/10/2020] [Accepted: 01/14/2020] [Indexed: 12/28/2022]
Abstract
G‐quadruplexes form folded structures because of tandem repeats of guanine sequences in DNA or RNA. They adopt a variety of conformations, depending on many factors, including the type of loops and cations, the nucleotide strand number, and the main strand polarity of the G‐quadruplex. Meanwhile, the different conformations of G‐quadruplexes have certain influences on their biological functions, such as the inhibition of transcription, translation, and DNA replication. In addition, G‐quadruplex binding proteins also affect the structure and function of G‐quadruplexes. Some chemically synthesized G‐quadruplex sequences have been shown to have biological activities. For example, bimolecular G‐quadruplexes of AS1411 act as targets of exogenous drugs that inhibit the proliferation of malignant tumours. G‐quadruplexes are also used as vehicles to deliver nanoparticles. Thus, it is important to identify the factors that influence G‐quadruplex structures and maintain the stability of G‐quadruplexes. Herein, we mainly discuss the factors influencing G‐quadruplexes and the synthetic G‐quadruplex, AS1411. Significance of the study This review summarizes the factors that influence G‐quadruplexes and the functions of the synthetic G‐quadruplex, AS1411. It also discusses the use of G‐quadruplexes for drug delivery in tumour therapy.
Collapse
Affiliation(s)
- Wen-Fang Yuan
- Medical College, China Three Gorges University, Yichang, China.,Institute of Organ Fibrosis and Targeted Drug Delivery, China Three Gorges University, Yichang, China.,Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
| | - Lin-Yan Wan
- The People's Hospital, China Three Gorges University, Yichang, China.,Institute of Organ Fibrosis and Targeted Drug Delivery, China Three Gorges University, Yichang, China
| | - Hu Peng
- Medical College, China Three Gorges University, Yichang, China.,Institute of Organ Fibrosis and Targeted Drug Delivery, China Three Gorges University, Yichang, China.,Surgeon, The Yiling Hospital of Yichang, Yichang, China.,Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
| | - Yuan-Mei Zhong
- Medical College, China Three Gorges University, Yichang, China
| | - Wen-Li Cai
- Medical College, China Three Gorges University, Yichang, China
| | - Yan-Qiong Zhang
- Medical College, China Three Gorges University, Yichang, China.,Institute of Organ Fibrosis and Targeted Drug Delivery, China Three Gorges University, Yichang, China.,Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
| | - Wen-Bing Ai
- Surgeon, The Yiling Hospital of Yichang, Yichang, China
| | - Jiang-Feng Wu
- Medical College, China Three Gorges University, Yichang, China.,The People's Hospital, China Three Gorges University, Yichang, China.,Institute of Organ Fibrosis and Targeted Drug Delivery, China Three Gorges University, Yichang, China.,Surgeon, The Yiling Hospital of Yichang, Yichang, China.,Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
| |
Collapse
|
|
45
|
Xie P. Non-tight and tight chemomechanical couplings of biomolecular motors under hindering loads. J Theor Biol 2020; 490:110173. [PMID: 31982418 DOI: 10.1016/j.jtbi.2020.110173] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2019] [Revised: 01/17/2020] [Accepted: 01/21/2020] [Indexed: 10/25/2022]
Abstract
Biomolecular motors make use of free energy released from chemical reaction (typically ATP hydrolysis) to perform mechanical motion or work. An important issue is whether a molecular motor exhibits tight or non-tight chemomechanical (CM) coupling. The tight CM coupling refers to that each ATPase activity is coupled with a mechanical step, while the non-tight CM coupling refers to that an ATPase activity is not necessarily coupled with a mechanical step. Here, we take kinesin, monomeric DNA helicase, ring-shaped hexameric DNA helicase and ribosome as examples to study this issue. Our studies indicate that some motors such as kinesin, monomeric helicase and ribosome exhibit non-tight CM coupling under hindering forces, while others such as the ring-shaped hexameric helicase exhibit tight or nearly tight CM coupling under any force. For the former, the reduction of the velocity caused by the hindering force arises mainly from the reduction of the CM coupling efficiency, while the ATPase rate is independent or nearly independent of the force. For the latter, the reduction of the velocity caused by the hindering force arises mainly from the reduction of the ATPase rate, while the CM coupling efficiency is independent or nearly independent of the force.
Collapse
Affiliation(s)
- Ping Xie
- Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.
| |
Collapse
|
|
46
|
Takahashi S, Sugimoto N. Stability prediction of canonical and non-canonical structures of nucleic acids in various molecular environments and cells. Chem Soc Rev 2020; 49:8439-8468. [DOI: 10.1039/d0cs00594k] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
This review provides the biophysicochemical background and recent advances in stability prediction of canonical and non-canonical structures of nucleic acids in various molecular environments and cells.
Collapse
Affiliation(s)
- Shuntaro Takahashi
- Frontier Institute for Biomolecular Engineering Research (FIBER)
- Konan University
- Kobe
- Japan
| | - Naoki Sugimoto
- Frontier Institute for Biomolecular Engineering Research (FIBER)
- Konan University
- Kobe
- Japan
- Graduate School of Frontiers of Innovative Research in Science and Technology (FIRST)
| |
Collapse
|
|
47
|
Lowran K, Campbell L, Popp P, Wu CG. Assembly of a G-Quadruplex Repair Complex by the FANCJ DNA Helicase and the REV1 Polymerase. Genes (Basel) 2019; 11:E5. [PMID: 31861576 PMCID: PMC7017153 DOI: 10.3390/genes11010005] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2019] [Accepted: 12/17/2019] [Indexed: 12/14/2022] Open
Abstract
The FANCJ helicase unfolds G-quadruplexes (G4s) in human cells to support DNA replication. This action is coupled to the recruitment of REV1 polymerase to synthesize DNA across from a guanine template. The precise mechanisms of these reactions remain unclear. While FANCJ binds to G4s with an AKKQ motif, it is not known whether this site recognizes damaged G4 structures. FANCJ also has a PIP-like (PCNA Interacting Protein) region that may recruit REV1 to G4s either directly or through interactions mediated by PCNA protein. In this work, we measured the affinities of a FANCJ AKKQ peptide for G4s formed by (TTAGGG)4 and (GGGT)4 using fluorescence spectroscopy and biolayer interferometry (BLI). The effects of 8-oxoguanine (8oxoG) on these interactions were tested at different positions. BLI assays were then performed with a FANCJ PIP to examine its recruitment of REV1 and PCNA. FANCJ AKKQ bound tightly to a TTA loop and was sequestered away from the 8oxoG. Reducing the loop length between guanine tetrads increased the affinity of the peptide for 8oxoG4s. FANCJ PIP targeted both REV1 and PCNA but favored interactions with the REV1 polymerase. The impact of these results on the remodeling of damaged G4 DNA is discussed herein.
Collapse
Affiliation(s)
- Kaitlin Lowran
- Department of Chemistry, Oakland University, Rochester, MI 48309, USA; (K.L.); (L.C.)
| | - Laura Campbell
- Department of Chemistry, Oakland University, Rochester, MI 48309, USA; (K.L.); (L.C.)
| | - Phillip Popp
- Case Western Reserve University, Cleveland, OH 44106, USA;
| | - Colin G. Wu
- Department of Chemistry, Oakland University, Rochester, MI 48309, USA; (K.L.); (L.C.)
| |
Collapse
|
|
48
|
Makurath MA, Whitley KD, Nguyen B, Lohman TM, Chemla YR. Regulation of Rep helicase unwinding by an auto-inhibitory subdomain. Nucleic Acids Res 2019; 47:2523-2532. [PMID: 30690484 PMCID: PMC6412110 DOI: 10.1093/nar/gkz023] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Revised: 12/26/2018] [Accepted: 01/16/2019] [Indexed: 11/29/2022] Open
Abstract
Helicases are biomolecular motors that unwind nucleic acids, and their regulation is essential for proper maintenance of genomic integrity. Escherichia coli Rep helicase, whose primary role is to help restart stalled replication, serves as a model for Superfamily I helicases. The activity of Rep-like helicases is regulated by two factors: their oligomeric state, and the conformation of the flexible subdomain 2B. However, the mechanism of control is not well understood. To understand the factors that regulate the active state of Rep, here we investigate the behavior of a 2B-deficient variant (RepΔ2B) in relation to wild-type Rep (wtRep). Using a single-molecule optical tweezers assay, we explore the effects of oligomeric state, DNA geometry, and duplex stability on wtRep and RepΔ2B unwinding activity. We find that monomeric RepΔ2B unwinds more processively and at a higher speed than the activated, dimeric form of wtRep. The unwinding processivity of RepΔ2B and wtRep is primarily limited by ‘strand-switching’—during which the helicases alternate between strands of the duplex—which does not require the 2B subdomain, contrary to a previous proposal. We provide a quantitative model of the factors that enhance unwinding processivity. Our work sheds light on the mechanisms of regulation of unwinding by Rep-like helicases.
Collapse
Affiliation(s)
- Monika A Makurath
- Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.,Department of Physics, Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Kevin D Whitley
- Department of Physics, Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.,Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Binh Nguyen
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Timothy M Lohman
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Yann R Chemla
- Department of Physics, Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| |
Collapse
|
|
49
|
Chakrabarti S, Jarzynski C, Thirumalai D. Processivity, Velocity, and Universal Characteristics of Nucleic Acid Unwinding by Helicases. Biophys J 2019; 117:867-879. [PMID: 31400912 PMCID: PMC6731385 DOI: 10.1016/j.bpj.2019.07.021] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2019] [Revised: 06/20/2019] [Accepted: 07/12/2019] [Indexed: 12/20/2022] Open
Abstract
Helicases are components of the cellular replisome that are essential for unwinding double-strand nucleic acids during the process of replication. Intriguingly, most helicases are inefficient and require either oligomerization or assistance from other partner proteins to increase the processivity of unwinding in the presence of the replication fork, which acts as a barrier to progress. Single-molecule force spectroscopy has emerged as a promising experimental technique to probe how relieving this barrier on the helicase can allow for increased efficiency of unwinding. However, there exists no comprehensive theoretical framework to provide unique interpretations of the underlying helicase kinetics from the force spectroscopy data. This remains a major confounding issue in the field. Here, we develop a mathematical framework and derive analytic expressions for the velocity and run length of a general model of finitely processive helicases, the two most commonly measured experimental quantities. We show that in contrast to the unwinding velocity, the processivity exhibits a universal increase in response to external force, irrespective of the underlying architecture and unwinding kinetics of the helicase. Our work provides the first, to our knowledge, explanation to a wide array of experiments and suggests that helicases may have evolved to maximize processivity rather than speed. To demonstrate the use of our theory on experimental data, we analyze velocity and processivity data on the T7 helicase and provide unique inferences on the kinetics of the helicase. Our results show that T7 is a weakly active helicase that destabilizes the fork ahead by less than 1 kBT and back steps very frequently while unwinding DNA. Our work generates fundamental insights into the force response of helicases and provides a widely applicable method for inferring the underlying helicase kinetics from force spectroscopy data.
Collapse
Affiliation(s)
- Shaon Chakrabarti
- Department of Data Sciences, Dana-Farber Cancer Institute, Boston, Massachusetts; Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, Massachusetts; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts.
| | - Christopher Jarzynski
- Department of Chemistry and Biochemistry, Institute for Physical Sciences and Technology, Department of Physics, University of Maryland, College Park, Maryland
| | - D Thirumalai
- Department of Chemistry, The University of Texas at Austin, Austin, Texas
| |
Collapse
|
|
50
|
Amiri H, Noller HF. A tandem active site model for the ribosomal helicase. FEBS Lett 2019; 593:1009-1019. [PMID: 30972734 DOI: 10.1002/1873-3468.13383] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2019] [Revised: 03/31/2019] [Accepted: 04/06/2019] [Indexed: 12/12/2022]
Abstract
During protein synthesis, the messenger RNA (mRNA) helicase activity of the ribosome ensures that codons are made single stranded before decoding. Here, based on recent structural and functional findings, a quantitative model is presented for a tandem arrangement of two helicase active sites on the ribosome. A distal site encounters mRNA structures first, one elongation cycle earlier than a proximal site. Although unwinding of encountered mRNA structures past the proximal site is required for translocation, two routes exist for translocation past the distal site: sliding, which requires unwinding, and stick-slip, which does not. The model accounts in detail for a number of findings related to the ribosomal helicase and provides a testable framework to further study mRNA unwinding.
Collapse
Affiliation(s)
- Hossein Amiri
- Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA.,Department of Molecular, Cell and Developmental Biology, Center for Molecular Biology of RNA, University of California at Santa Cruz, Santa Cruz, CA, USA
| | - Harry F Noller
- Department of Molecular, Cell and Developmental Biology, Center for Molecular Biology of RNA, University of California at Santa Cruz, Santa Cruz, CA, USA
| |
Collapse
|