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O' Lee DJ, Wynveen A, Albrecht T, Kornyshev AA. Which way up? Recognition of homologous DNA segments in parallel and antiparallel alignments. J Chem Phys 2015; 142:045101. [PMID: 25638008 DOI: 10.1063/1.4905291] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Homologous gene shuffling between DNA molecules promotes genetic diversity and is an important pathway for DNA repair. For this to occur, homologous genes need to find and recognize each other. However, despite its central role in homologous recombination, the mechanism of homology recognition has remained an unsolved puzzle of molecular biology. While specific proteins are known to play a role at later stages of recombination, an initial coarse grained recognition step has, however, been proposed. This relies on the sequence dependence of the DNA structural parameters, such as twist and rise, mediated by intermolecular interactions, in particular, electrostatic ones. In this proposed mechanism, sequences that have the same base pair text, or are homologous, have lower interaction energy than those sequences with uncorrelated base pair texts. The difference between the two energies is termed the "recognition energy." Here, we probe how the recognition energy changes when one DNA fragment slides past another, and consider, for the first time, homologous sequences in antiparallel alignment. This dependence on sliding is termed the "recognition well." We find there is a recognition well for anti-parallel, homologous DNA tracts, but only a very shallow one, so that their interaction will differ little from the interaction between two nonhomologous tracts. This fact may be utilized in single molecule experiments specially targeted to test the theory. As well as this, we test previous theoretical approximations in calculating the recognition well for parallel molecules against MC simulations and consider more rigorously the optimization of the orientations of the fragments about their long axes upon calculating these recognition energies. The more rigorous treatment affects the recognition energy a little, when the molecules are considered rigid. When torsional flexibility of the DNA molecules is introduced, we find excellent agreement between the analytical approximation and simulations.
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Affiliation(s)
- Dominic J O' Lee
- Department of Chemistry, Imperial College London, SW7 2AZ London, United Kingdom
| | - Aaron Wynveen
- School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota 55455, USA
| | - Tim Albrecht
- Department of Chemistry, Imperial College London, SW7 2AZ London, United Kingdom
| | - Alexei A Kornyshev
- Department of Chemistry, Imperial College London, SW7 2AZ London, United Kingdom
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De-convoluting the Genetic Adaptations of E. coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields. Cell Rep 2015; 10:1758-1766. [DOI: 10.1016/j.celrep.2015.02.029] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2014] [Revised: 01/29/2015] [Accepted: 02/08/2015] [Indexed: 11/20/2022] Open
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Brimacombe CA, Ding H, Beatty JT. Rhodobacter capsulatus DprA is essential for RecA-mediated gene transfer agent (RcGTA) recipient capability regulated by quorum-sensing and the CtrA response regulator. Mol Microbiol 2014; 92:1260-78. [DOI: 10.1111/mmi.12628] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/24/2014] [Indexed: 01/18/2023]
Affiliation(s)
- Cedric A. Brimacombe
- Department of Microbiology and Immunology; The University of British Columbia; 2350 Health Sciences Mall Vancouver BC Canada V6T 1Z3
| | - Hao Ding
- Department of Microbiology and Immunology; The University of British Columbia; 2350 Health Sciences Mall Vancouver BC Canada V6T 1Z3
| | - J. Thomas Beatty
- Department of Microbiology and Immunology; The University of British Columbia; 2350 Health Sciences Mall Vancouver BC Canada V6T 1Z3
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Mair RW, Senadheera DB, Cvitkovitch DG. CinA is regulated via ComX to modulate genetic transformation and cell viability in Streptococcus mutans. FEMS Microbiol Lett 2012; 331:44-52. [PMID: 22428842 DOI: 10.1111/j.1574-6968.2012.02550.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2011] [Revised: 03/08/2012] [Accepted: 03/13/2012] [Indexed: 11/29/2022] Open
Abstract
The Streptococcus mutans ComX-regulon encompasses > 200 mostly uncharacterized genes, including cinA. Here we report that cinA is regulated by ComX in the presence of the competence stimulating peptide (CSP), wherein loss of CinA (strain SmuCinA) results in reduced transformability with or without added CSP by 74- and 15-fold, respectively (P < 0.003). In CSP-supplemented cultures, a two-fold increase in cell viability was noted for SmuCinA relative to UA159 (P < 0.002), suggesting CinA's involvement in the CSP-modulated cell killing response. Relative to UA159, loss of CinA also rendered the mutant hypersensitive to killing by methyl methanesulfonate (MMS), which impairs homologous recombination. Despite our use of a non-polar mutagenesis strategy to knockout cinA, which is the first gene of the multicistronic operon harboring cinA, we noted a drastic reduction in recA expression. By using a CinA-complemented mutant, we were able to partially, but not completely restore all phenotypes to UA159 levels. Complementation results suggested that although cinA participates in modulating competence, viability and MMS tolerance, genes downstream of the cinA transcript may also regulate these phenotypes, a finding that warrants further examination. This is the first report that describes a role for S. mutans' CinA in contending with DNA damage, genetic transformation and cell survival.
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Affiliation(s)
- Richard W Mair
- Dental Research Institute, University of Toronto, Toronto, ON, Canada
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Kornyshev AA. Physics of DNA: unravelling hidden abilities encoded in the structure of ‘the most important molecule’. Phys Chem Chem Phys 2011; 12:12352-78. [PMID: 20945523 DOI: 10.1039/c004107f] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
A comprehensive article “Structure and Interactions of Biological Helices”, published in 2007 in Reviews of Modern Physics, overviewed various aspects of the effect of DNA structure on DNA–DNA interactions in solution and related phenomena, with a thorough analysis of the theory of these effects. Here, an updated qualitative account of this area is presented without any sophisticated ‘algebra’. It overviews the basic principles of the structure-specific interactions between double-stranded DNA and focuses on the physics behind several related properties encoded in the structure of DNA. Among them are (i) DNA condensation and aptitude to pack into small compartments of cells or viral capcids, (ii) the structure of DNA mesophases, and (iii) the ability of homologous genes to recognize each other prior to recombination from a distance. Highlighted are some of latest developments of the theory, including the shape of the ‘recognition well’. The article ends with a brief discussion of the first experimental evidence of the protein-free homology recognition in a ‘test tube’.
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Affiliation(s)
- Alexei A Kornyshev
- Department of Chemistry, Faculty of Natural Sciences, South Kensington Campus, Imperial College London, SW7 2AZ, UK.
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6
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Abstract
The recognition of homologous sequences of DNA before strand exchange is considered to be the most puzzling stage of homologous recombination. A mechanism for two homologous dsDNAs to recognize each other from a distance in electrolytic solution without unzipping had been proposed in an earlier paper [A. A. Kornyshev and S. Leikin, Phys. Rev. Lett. 86, 366 (2001)]. In that work, the difference in the electrostatic interaction energy between homologous duplexes and between nonhomologous duplexes, termed the recognition energy, has been calculated. That calculation was later extended in a series of papers to account for torsional elasticity of the molecules. A recent paper [A. A. Kornyshev and A. Wynveen, Proc. Natl. Acad. Sci. U.S.A. 106, 4683 (2009)] investigated the form of the potential well that homologous DNA molecules may feel when sliding along each other. A simple formula for the shape of the well was obtained. However, this latter study was performed under the approximation that the sliding molecules are torsionally rigid. Following on from this work, in the present article we investigate the effect of torsional flexibility of the molecules on the shape of the well. A variational approach to this problem results in a transcendental equation that is easily solved numerically. Its solutions show that at large interaxial separations the recognition well becomes wider and shallower, whereas at closer distances further unexpected features arise related to an abrupt change in the mean azimuthal alignment of the molecules. The energy surface as a function of interaxial separation and the axial shift defines what we call the recognition funnel. We show that it depends dramatically on the patterns of adsorption of counterions on DNA.
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Affiliation(s)
- Dominic Lee
- Max Planck Institute for the Physics of Complex Systems, D-01187 Dresden, Germany.
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7
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Cherstvy AG. DNA-DNA sequence homology recognition: physical mechanisms and open questions. J Mol Recognit 2010; 24:283-7. [DOI: 10.1002/jmr.1050] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2010] [Revised: 04/07/2010] [Accepted: 04/10/2010] [Indexed: 11/11/2022]
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Yamamoto S, Izumiya H, Morita M, Arakawa E, Watanabe H. Application of λ Red recombination system to Vibrio cholerae genetics: Simple methods for inactivation and modification of chromosomal genes. Gene 2009; 438:57-64. [DOI: 10.1016/j.gene.2009.02.015] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2009] [Revised: 02/24/2009] [Accepted: 02/25/2009] [Indexed: 12/26/2022]
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Prabu JR, Thamotharan S, Khanduja JS, Chandra NR, Muniyappa K, Vijayan M. Crystallographic and modelling studies on Mycobacterium tuberculosis RuvA Additional role of RuvB-binding domain and inter species variability. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2009; 1794:1001-9. [PMID: 19374958 DOI: 10.1016/j.bbapap.2009.04.003] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/26/2008] [Revised: 04/06/2009] [Accepted: 04/06/2009] [Indexed: 11/28/2022]
Abstract
RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of three new crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures presented here and those reported earlier brings to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in Holliday junction binding. This role along with its role in oligomerization could have important biological implications.
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Affiliation(s)
- J Rajan Prabu
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India
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10
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Lutz KA, Maliga P. Construction of marker-free transplastomic plants. Curr Opin Biotechnol 2007; 18:107-14. [PMID: 17339108 DOI: 10.1016/j.copbio.2007.02.003] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2007] [Revised: 02/05/2007] [Accepted: 02/23/2007] [Indexed: 11/17/2022]
Abstract
Because of its prokaryotic-type gene expression machinery, maternal inheritance and the opportunity to express proteins at a high level, the plastid genome (plastome or ptDNA) is an increasingly popular target for engineering. The ptDNA is present as up to 10,000 copies per cell, making selection for marker genes essential to obtain plants with uniformly transformed ptDNA. However, the marker gene is no longer desirable when homoplastomic plants are obtained. Marker-free transplastomic plants can now be obtained with four recently developed protocols: homology-based excision via directly repeated sequences, excision by phage site-specific recombinanses, transient cointegration of the marker gene, and the cotransformation-segregation approach. Marker excision technology will benefit applications in agriculture and in molecular farming.
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Affiliation(s)
- Kerry A Lutz
- Waksman Institute, Rutgers, State University of New Jersey, Piscataway, NJ 08854-8020, USA
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11
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Drury MD, Kmiec EB. Double displacement loops (double d-loops) are templates for oligonucleotide-directed mutagenesis and gene repair. Oligonucleotides 2005; 14:274-86. [PMID: 15665595 DOI: 10.1089/oli.2004.14.274] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Appreciable levels of gene repair result from the hybridization of two oligonucleotides at a specific site in a mutated gene and subsequent correction by a form of oligonucleotide-directed mutagenesis known as gene repair. The incorporation of the two oligonucleotides into superhelical plasmid DNA leads to the formation of double d-loops, structures shown to be templates for the repair of both frameshift and point mutations. Structural limitations placed on the template indicate that correction is influenced significantly by the positioning of the second oligonucleotide, known as the annealing oligonucleotide. Complexes constructed with two oligonucleotides directly opposite each other exhibit the highest levels of gene repair activity. Blocking the 3'-end of either oligonucleotide with an amino C7 group does not diminish the performance of the double d-loop as a template for correction of the point mutation, suggesting that primer extension does not play a pivotal role in the mechanism of gene repair.
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Affiliation(s)
- Miya D Drury
- Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA
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12
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Chen LM, Le HY, Qin RY, Kumar M, Du ZY, Xia RJ, Deng J. Reversal of the phenotype by K-rasval12 silencing mediated by adenovirus-delivered siRNA in human pancreatic cancer cell line Panc-1. World J Gastroenterol 2005; 11:831-8. [PMID: 15682475 PMCID: PMC4250591 DOI: 10.3748/wjg.v11.i6.831] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the in vitro antitumor effect of adenovirus-mediated small interfering RNAs (siRNAs) on pancreatic cancer and the associated mechanism.
METHODS: A 63-nucleotide (nt) oligonucleotide encoding K-rasval12 and specific siRNA were introduced into pSilencer 3.1-H1, then the H1-RNA promoter and siRNA coding insert were subcloned into pAdTrack to get plasmid pAdTrackH1-K-rasval12. After homologous recombination in bacteria and transfections of such plasmids into a mammalian packaging cell line 293, siRNA expressing adenovirus AdH1-K-rasval12 was obtained. Stable suppression of K-rasval12 was detected by Northern blot and Western blot. Apoptosis in Panc-1 cells was detected by flow cytometry.
RESULTS: We obtained adenovirus AdH1-K-rasval12 carrying the pSilencer 3.1-H1 cassette, which could mediate gene silencing. Through siRNA targeted K-rasval12, the oncogenic phenotype of cancer cells was reversed. Flow cytometry showed that apoptotic index of Panc-1 cells was significantly higher in the AdH1-K-rasval12-treatment group (18.70% at 72 h post-infection, 49.55% at 96 h post-infection) compared to the control groups (3.47%, 3.98% at 72 and 96 h post-infection of AdH1-empty, respectively; 4.21%, 3.78% at 72 and 96 h post-infection of AdH1-p53, respectively) (P<0.05).
CONCLUSION: These results demonstrate that adenoviral vectors can be used to mediate RNA interference (RNAi) to induce persistent loss of functional phenotypes. In gene therapy, the selective down-regulation of only the mutant version of a gene allows for highly specific effects on tumor cells, while leaving the normal cells untouched. In addition, the apoptosis of pancreatic cancer cell line Panc-1 can be induced after AdH1-K-rasval12 infection. This kind of adenovirus based on RNAi might be a promising vector for cancer therapy.
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Affiliation(s)
- Li-Mo Chen
- Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
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Cellini F, Chesson A, Colquhoun I, Constable A, Davies HV, Engel KH, Gatehouse AMR, Kärenlampi S, Kok EJ, Leguay JJ, Lehesranta S, Noteborn HPJM, Pedersen J, Smith M. Unintended effects and their detection in genetically modified crops. Food Chem Toxicol 2004; 42:1089-125. [PMID: 15123383 DOI: 10.1016/j.fct.2004.02.003] [Citation(s) in RCA: 198] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2003] [Accepted: 02/04/2004] [Indexed: 11/28/2022]
Abstract
The commercialisation of GM crops in Europe is practically non-existent at the present time. The European Commission has instigated changes to the regulatory process to address the concerns of consumers and member states and to pave the way for removing the current moratorium. With regard to the safety of GM crops and products, the current risk assessment process pays particular attention to potential adverse effects on human and animal health and the environment. This document deals with the concept of unintended effects in GM crops and products, i.e. effects that go beyond that of the original modification and that might impact primarily on health. The document first deals with the potential for unintended effects caused by the processes of transgene insertion (DNA rearrangements) and makes comparisons with genetic recombination events and DNA rearrangements in traditional breeding. The document then focuses on the potential value of evolving "profiling" or "omics" technologies as non-targeted, unbiased approaches, to detect unintended effects. These technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). The technologies are described, together with their current limitations. Importantly, the significance of unintended effects on consumer health are discussed and conclusions and recommendations presented on the various approaches outlined.
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Affiliation(s)
- F Cellini
- Metapontum Agrobios, SS Jonica Km 448.2, I-75010 Metaponto Matera, Italy
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14
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Critchlow HM, Payne A, Griffin DK. Genes and proteins involved in the control of meiosis. Cytogenet Genome Res 2004; 105:4-10. [PMID: 15218251 DOI: 10.1159/000078002] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2003] [Accepted: 11/04/2003] [Indexed: 11/19/2022] Open
Affiliation(s)
- H M Critchlow
- Cell and Chromosome Biology Group, Department of Biological Sciences, Brunel University, Uxbridge, UK
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Dennis C, Fedorov A, Käs E, Salomé L, Grigoriev M. RuvAB-directed branch migration of individual Holliday junctions is impeded by sequence heterology. EMBO J 2004; 23:2413-22. [PMID: 15167893 PMCID: PMC423290 DOI: 10.1038/sj.emboj.7600249] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2003] [Accepted: 04/29/2004] [Indexed: 11/09/2022] Open
Abstract
The Holliday junction, the key intermediate of recombination, is generated by strand exchange resulting in a covalent connection between two recombining DNA molecules. Translocation of a Holliday junction along DNA, or branch migration, progressively exchanges one DNA strand for another and determines the amount of information that is transferred between two recombining partners. In Escherichia coli, the RuvAB protein complex promotes rapid and unidirectional branch migration of Holliday junctions. We have studied translocation of Holliday junctions using a quantitative biochemical system together with a 'single-molecule' branch migration assay. We demonstrate that RuvAB translocates the junctions through identical DNA sequences in a processive manner with a broad distribution of individual branch migration rates. However, when the complex encounters short heterologous sequences, translocation of the Holliday junctions is impeded. We conclude that translocation of the junctions through a sequence heterology occurs with a probability of bypass being determined both by the length of the heterologous region and the lifetime of the stalled RuvAB complex.
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Affiliation(s)
- Cynthia Dennis
- Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS and Université Paul Sabatier, Toulouse, France
| | - Andrei Fedorov
- Centre d'Etude Spatiale des Rayonnements, UPR 8002 CNRS, Toulouse, France
| | - Emmanuel Käs
- Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS and Université Paul Sabatier, Toulouse, France
| | - Laurence Salomé
- Institut de Pharmacologie et Biologie Structurale, UMR 5089 CNRS, Toulouse, France
| | - Mikhail Grigoriev
- Laboratoire de Biologie Moléculaire Eucaryote, UMR 5099 CNRS and Université Paul Sabatier, Toulouse, France
- Laboratoire de Biologie Mol Eucaryote, UMR 5099 CNRS, Institut d'Exploration Fonctionelle des Génomes, 118 route de Narbonne, 31062 Toulouse, France. Tel.: +33 5 61 33 58 08; Fax: +33 5 61 33 58 86; E-mail:
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Yamazaki J, Horii T, Sekiguchi M, Takahashi M. Regulation of RecA protein binding to DNA by opposing effects of ATP and ADP on inter-domain contacts: analysis by urea-induced unfolding of wild-type and C-terminal truncated RecA. J Mol Biol 2003; 329:363-70. [PMID: 12758082 DOI: 10.1016/s0022-2836(03)00449-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
RecA protein requires ATP and its hydrolysis to ADP to complete the DNA strand-exchange reaction. We investigated how the nucleotides activate RecA by examining their effect on urea-induced unfolding, which could reflect domain-domain contact of protein. RecA is folded into three continuous domains: the N-terminal, central and C-terminal domains. The fluorescence of tyrosine residues, which lie mainly in the central domain, was modified in 1-3 M urea, while the red shift of fluorescence peak of the tryptophan residues located in the C-terminal domain occurred only in 3-6 M urea. Thus, the C-terminal domain of RecA is unfolded after the central part unfolds. The change in intensity of tryptophan fluorescence without a large shift in the peak at low concentrations of urea suggests that there are weak interactions between the central and C-terminal domains. This is supported by our observation that RecA protein lacking the C-terminal tail unfolded at lower concentrations of urea than the entire RecA, and with clear transitions, unlike the entire RecA. ATP and its unhydrolyzable analog (ATPgammaS), which enhance the binding of RecA to DNA, facilitated the urea-induced change in RecA tryptophan fluorescence, while ADP, an antagonist of ATP, prevented the change. ATP probably weakens the domain-domain contact and facilitates the DNA binding, while ADP stabilizes the contact and inhibits it. Supporting this conclusion, the binding of RecA lacking the C-terminal tail to DNA was not inhibited by ADP, while that of the intact RecA was.
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Affiliation(s)
- Jun Yamazaki
- Frontier Research Centre and Fukuoka Dental College, Japan
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Dutreix M, Fulconis R, Viovy JL. The Search for Homology: A Paradigm for Molecular Interactions? ACTA ACUST UNITED AC 2003. [DOI: 10.1159/000070465] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
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Reiss B. Homologous recombination and gene targeting in plant cells. INTERNATIONAL REVIEW OF CYTOLOGY 2003; 228:85-139. [PMID: 14667043 DOI: 10.1016/s0074-7696(03)28003-7] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Gene targeting has become an indispensable tool for functional genomics in yeast and mouse; however, this tool is still missing in plants. This review discusses the gene targeting problem in plants in the context of general knowledge on recombination and gene targeting. An overview on the history of gene targeting is followed by a general introduction to genetic recombination of bacteria, yeast, and vertebrates. This abridged discussion serves as a guide to the following sections, which cover plant-specific aspects of recombination assay systems, the mechanism of recombination, plant recombination genes, the relationship of recombination to the environment, approaches to stimulate homologous recombination and gene targeting, and a description of two plant systems, the moss Physcomitrella patens and the chloroplast, that naturally have high efficiencies of gene targeting. The review concludes with a discussion of alternatives to gene targeting.
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Affiliation(s)
- Bernd Reiss
- Max-Planck-Institut für Zuechtungsforschung, Carl-von-Linne-Weg 10, D-50829 Köln, Germany
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Sekkai D, Dausse E, Di Primo C, Darfeuille F, Boiziau C, Toulmé JJ. In vitro selection of DNA aptamers against the HIV-1 TAR RNA hairpin. ANTISENSE & NUCLEIC ACID DRUG DEVELOPMENT 2002; 12:265-74. [PMID: 12238815 DOI: 10.1089/108729002320351584] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
In vitro selection was performed to identify DNA aptamers against the TAR RNA stem-loop structure of HIV-1. A counterselection step allowed the elimination of kissing complex-forming aptamers previously selected (Boiziau et al. J. Biol. Chem. 1999; 274:12730). This led to the emergence of oligonucleotides, most of which contained two consensus sequences, one targeted to the stem 3'-strand (5'-CCCTAGTTA) and the other complementary to the TAR apical loop (5'-CTCCC). The best aptamer could be shortened to a 19-mer oligonucleotide, characterized by a dissociation constant of 50 nM. A 16-mer oligonucleotide complementary to the TAR stem 3'-strand could also be derived from the identified aptamers, with an equal affinity (Kd = 50 nM). Experiments performed to elucidate the interaction between TAR and the aptamers (UV melting measures, enzymatic and chemical footprints) demonstrated that the TAR stem 5'-strand was not simply displaced as a result of the complex formation but unexpectedly remained associated on contact with the antisense oligonucleotide. We suggest that a multistranded structure could be formed.
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Affiliation(s)
- Dalila Sekkai
- INSERM U 386, Université Victor Segalen Bordeaux 2, France
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Xiao J, Singleton SF. Elucidating a key intermediate in homologous DNA strand exchange: structural characterization of the RecA-triple-stranded DNA complex using fluorescence resonance energy transfer. J Mol Biol 2002; 320:529-58. [PMID: 12096908 DOI: 10.1016/s0022-2836(02)00462-x] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA-tsDNA complex), has been reported. We present a systematic characterization of the helical geometries of the three DNA strands of the RecA-tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. FRET donor and acceptor dyes were used to label different DNA strands, and the interfluorophore distances were inferred from energy transfer efficiencies measured as a function of the base-pair separation between the two dyes. The energy transfer efficiencies were first measured on a control RecA-dsDNA complex, and the calculated helical parameters (h approximately 5 A, Omega(h) approximately 20 degrees ) were consistent with structural conclusions derived from electron microscopy (EM) and other classic biochemical methods. Measurements of the helical parameters for the RecA-tsDNA complex revealed that all three DNA strands adopt extended and unwound conformations similar to those of RecA-bound dsDNA. The structural data are consistent with the hypothesis that this complex is a late, post-strand-exchange intermediate with the outgoing strand shifted by about three base-pairs with respect to its registry with the incoming and complementary strands. Furthermore, the bases of the incoming and complementary strands are displaced away from the helix axis toward the minor groove of the heteroduplex, and the bases of the outgoing strand lie in the major groove of the heteroduplex. We present a model for the strand exchange intermediate in which homologous contacts preceding strand exchange arise in the minor groove of the substrate dsDNA.
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Affiliation(s)
- Jie Xiao
- Department of Chemistry, Rice University, P.O. Box 1892, MS 65, Houston, TX 77005, USA
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21
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Ayora S, Piruat JI, Luna R, Reiss B, Russo VEA, Aguilera A, Alonso JC. Characterization of two highly similar Rad51 homologs of Physcomitrella patens. J Mol Biol 2002; 316:35-49. [PMID: 11829501 DOI: 10.1006/jmbi.2001.5336] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The moss Physcomitrella patens, which is a land plant with efficient homologous recombination, encodes two Rad51 proteins (PpaRad51.1 and PpaRad51.2). The PpaRad51.1 and PpaRad51.2 proteins, which share 94 % identity between them, interact with themselves and with each other. Both proteins bind ssDNA and dsDNA in a Mg(2+) and pH-dependent manner, with a stoichiometry of one PpaRad51.1 monomer per 3(+/-1) nt or bp and one PpaRad51.2 monomer per 1(+/-0.5) nt or bp, respectively. At neutral pH, a 1.6-fold excess of both proteins is required for ssDNA and dsDNA binding. PpaRad51.1 and PpaRad51.2 show ssDNA-dependent ATPase activity and efficiently promote strand annealing in a nucleotide-independent but in a Mg(2+)-dependent manner. Both proteins promote joint-molecule formation, DNA strand invasion and are able to catalyse strand exchange in the presence of Mg(2+) and ATP. No further increase in the activities is observed when both proteins are present in the same reaction. None of the PpaRad51 gene products complement the DNA repair and recombination phenotype of Saccharomyces cerevisiae rad51delta mutants. However, PpaRad51.1 confers a dominant-negative DNA repair phenotype, and both PpaRad51 proteins reduce the levels of double-strand break-induced recombination when overexpressed in S. cerevisiae wt cells. These results suggest that both PpaRad51 proteins are bona fide Rad51 proteins that may contribute, in a different manner, to homologous recombination, and that they might replace ScRad51 in a hypothetical yeast protein complex inactivating different functions required for recombinational repair.
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Affiliation(s)
- Silvia Ayora
- Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Cantoblanco, Campus Universidad Autónoma de Madrid, 28049, Spain
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22
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Zhang Z, Yoon D, LaPorte JR, Chen J. Appropriate initiation of the strand exchange reaction promoted by RecA protein requires ATP hydrolysis. J Mol Biol 2001; 309:29-43. [PMID: 11491297 DOI: 10.1006/jmbi.2001.4753] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The DNA-dependent ATPase activity of the Escherichia coli RecA protein has been recognized for more than two decades. Yet, the role of ATP hydrolysis in the RecA-promoted strand exchange reaction remains unclear. Here, we demonstrate that ATP hydrolysis is required as part of a proofreading process during homology recognition. It enables the RecA-ssDNA complex, after determining that the strand-exchanged duplex is mismatched, to dissociate from the synaptic complex, which allows it to re-initiate the search for a "true" homologous region. Furthermore, the results suggest that when non-homologous sequences are present at the proximal end, ATP hydrolysis is required to allow ssDNA-RecA to reinitiate the strand exchange from an internal homologous region.
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Affiliation(s)
- Z Zhang
- Department of Chemistry & Biochemistry, University of Delaware, Newark 19716, USA
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23
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Kornyshev AA, Leikin S. Sequence recognition in the pairing of DNA duplexes. PHYSICAL REVIEW LETTERS 2001; 86:3666-3669. [PMID: 11328049 DOI: 10.1103/physrevlett.86.3666] [Citation(s) in RCA: 71] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/20/2000] [Indexed: 05/23/2023]
Abstract
Pairing of DNA fragments with homologous sequences occurs in gene shuffling, DNA repair, and other vital processes. While chemical individuality of base pairs is hidden inside the double helix, x ray and NMR revealed sequence-dependent modulation of the structure of DNA backbone. Here we show that the resulting modulation of the DNA surface charge pattern enables duplexes longer than approximately 50 base pairs to recognize sequence homology electrostatically at a distance of up to several water layers. This may explain the local recognition observed in pairing of homologous chromosomes and the observed length dependence of homologous recombination.
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24
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Mazin AV, Bornarth CJ, Solinger JA, Heyer WD, Kowalczykowski SC. Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament. Mol Cell 2000; 6:583-92. [PMID: 11030338 DOI: 10.1016/s1097-2765(00)00057-5] [Citation(s) in RCA: 150] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Rad51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. Rad51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Here, we demonstrate that Rad54 acts in concert with the mature Rad51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to Rad51 within the filament. Reciprocally, the Rad51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. We conclude that Rad54 participates in the DNA homology search as a component of the Rad51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process.
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Affiliation(s)
- A V Mazin
- Division of Biological Sciences, University of California, Davis 95616, USA
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25
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Abstract
The budding yeast Saccharomyces cerevisiae has been an excellent genetic and biochemical model for our understanding of homologous recombination. Central to the process of homologous recombination are the products of the RAD52 epistasis group of genes, whose functions we now know include the nucleolytic processing of DNA double-stand breaks, the ability to conduct a DNA homology search, and the capacity to promote the exchange of genetic information between homologous regions on recombining chromosomes. It is also clear that the basic functions of the RAD52 group of genes have been highly conserved among eukaryotes. Disruption of this important process causes genomic instability, which can result in a number of unsavory consequences, including tumorigenesis and cell death.
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Affiliation(s)
- P Sung
- Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA.
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26
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Abstract
The ability to recognize and repair abnormal DNA structures is common to all forms of life. Studies in a variety of species have identified an incredible diversity of DNA repair pathways. Documenting and characterizing the similarities and differences in repair between species has important value for understanding the origin and evolution of repair pathways as well as for improving our understanding of phenotypes affected by repair (e.g., mutation rates, lifespan, tumorigenesis, survival in extreme environments). Unfortunately, while repair processes have been studied in quite a few species, the ecological and evolutionary diversity of such studies has been limited. Complete genome sequences can provide potential sources of new information about repair in different species. In this paper, we present a global comparative analysis of DNA repair proteins and processes based upon the analysis of available complete genome sequences. We use a new form of analysis that combines genome sequence information and phylogenetic studies into a composite analysis we refer to as phylogenomics. We use this phylogenomic analysis to study the evolution of repair proteins and processes and to predict the repair phenotypes of those species for which we now know the complete genome sequence.
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Affiliation(s)
- J A Eisen
- Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA.
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27
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Bernstein C, Bernstein H, Payne C. Cell immortality: maintenance of cell division potential. PROGRESS IN MOLECULAR AND SUBCELLULAR BIOLOGY 1999; 24:23-50. [PMID: 10547857 DOI: 10.1007/978-3-662-06227-2_2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Affiliation(s)
- C Bernstein
- Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson 85724, USA
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28
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Gupta RC, Folta-Stogniew E, O'Malley S, Takahashi M, Radding CM. Rapid exchange of A:T base pairs is essential for recognition of DNA homology by human Rad51 recombination protein. Mol Cell 1999; 4:705-14. [PMID: 10619018 DOI: 10.1016/s1097-2765(00)80381-0] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Human Rad51 belongs to a ubiquitous family of proteins that enable a single strand to recognize homology in duplex DNA, and thereby to initiate genetic exchanges and DNA repair, but the mechanism of recognition remains unknown. Kinetic analysis by fluorescence resonance energy transfer combined with the study of base substitutions and base mismatches reveals that recognition of homology, helix destabilization, exchange of base pairs, and initiation of strand exchange are integral parts of a rapid, concerted mechanism in which A:T base pairs play a critical role. Exchange of base pairs is essential for recognition of homology, and physical evidence indicates that such an exchange occurs early enough to mediate recognition.
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Affiliation(s)
- R C Gupta
- Department of Genetics, Yale University, New Haven, Connecticut 06510, USA
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29
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Bertolla F, Simonet P. Horizontal gene transfers in the environment: natural transformation as a putative process for gene transfers between transgenic plants and microorganisms. Res Microbiol 1999; 150:375-84. [PMID: 10466405 DOI: 10.1016/s0923-2508(99)80072-2] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Horizontal gene transfers among bacteria, such as natural transformation or conjugation, may have played an important role in bacterial evolution. They are thought to have been involved in promoting genome plasticity which permitted bacteria to adapt very efficiently to any change in their environment and to colonize a wide range of ecosystems. Evidence that some genes were transferred from eukaryotes, and in particular, from plants to bacteria, was obtained from nucleotide and protein sequence analyses. However, numerous factors, including some which are endogenous to the bacterial cells, tend to limit the extent of transfer, particularly among phylogenetically distant organisms. The goal of this paper is to give an overview of the potentials and limits of natural interkingdom gene transfers, with particular focus on prokaryote-originating sequences which fit the nuclear genome of transgenic plants.
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Affiliation(s)
- F Bertolla
- Laboratoire d'Ecologie Microbienne du Sol, UMR CNRS 5557, Université Lyon I, Villeurbanne, France.
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30
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Grigoriev M, Hsieh P. Migration of a Holliday junction through a nucleosome directed by the E. coli RuvAB motor protein. Mol Cell 1998; 2:373-81. [PMID: 9774975 DOI: 10.1016/s1097-2765(00)80281-6] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Chromatin plays a critical role in regulating access to DNA by proteins that direct recombination and repair. The E. coli RuvAB protein complex promotes branch migration of the Holliday junction recombination intermediate. The ability of RuvAB to negotiate passage of the junction through nucleosomal DNA is examined. The model system involves the formation of a Holliday junction positioned upstream of a nucleosome. Unassisted, the junction is blocked by a histone octamer. In the presence of RuvAB and ATP, rapid branch migration through the nucleosome is observed. It results in disruption of the histone-DNA interactions leading to the removal of the octamer from the junction intermediate. These results suggest that eukaryotic DNA motor proteins analogous to RuvAB could function during recombination to promote branch migration through chromatin.
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Affiliation(s)
- M Grigoriev
- Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1810, USA
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31
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Wang L, Voloshin ON, Stasiak A, Camerini-Otero RD. Homologous DNA pairing domain peptides of RecA protein: intrinsic propensity to form beta-structures and filaments. J Mol Biol 1998; 277:1-11. [PMID: 9514744 DOI: 10.1006/jmbi.1997.1591] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The 20 amino acid residue peptides derived from RecA loop L2 have been shown to be the pairing domain of RecA. The peptides bind to ss- and dsDNA, unstack ssDNA, and pair the ssDNA to its homologous target in a duplex DNA. As shown by circular dichroism, upon binding to DNA the disordered peptides adopt a beta-structure conformation. Here we show that the conformational change of the peptide from random coil to beta-structure is important in binding ss- and dsDNA. The beta-structure in the DNA pairing peptides can be induced by many environmental conditions such as high pH, high concentration, and non-micellar sodium dodecyl sulfate (6 mM). This behavior indicates an intrinsic property of these peptides to form a beta-structure. A beta-structure model for the loop L2 of RecA protein when bound to DNA is thus proposed. The fact that aromatic residues at the central position 203 strongly modulate the peptide binding to DNA and subsequent biochemical activities can be accounted for by the direct effect of the aromatic amino acids on the peptide conformational change. The DNA-pairing domain of RecA visualized by electron microscopy self-assembles into a filamentous structure like RecA. The relevance of such a peptide filamentous structure to the structure of RecA when bound to DNA is discussed.
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Affiliation(s)
- L Wang
- Rm 9D20, National Institutes of Health, Bethesda, MD 20892, USA
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32
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Jung KH, Lee DH, Yoon KH, Park SH. Integration and amplification of the Bacillus sp. 79-23 cellulase gene in the Bacillus subtilis 168 chromosome. J GEN APPL MICROBIOL 1998; 44:107-111. [PMID: 12501300 DOI: 10.2323/jgam.44.107] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Affiliation(s)
- Kyung-Hwa Jung
- Bacterial Molecular Genetics Research Unit, Korea Research Institute of Bioscience & Biotechnology (KRIBB), P.O. Box 115, Yusong, Taejon 305-600, Korea
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33
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Kannouche P, Pinon-Lataillade G, Mauffrey P, Faucher C, Biard DS, Angulo JF. Overexpression of kin17 protein forms intranuclear foci in mammalian cells. Biochimie 1997; 79:599-606. [PMID: 9466698 DOI: 10.1016/s0300-9084(97)82009-3] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
We used antibodies against E coli RecA protein to identify in mouse cells a 45-kDa DNA-binding protein called kin17, which has an active zinc finger and a nuclear localisation signal. Kin17 protein produced in E coli binds preferentially to the curved DNA of a bacterial promoter in vivo and in vitro, suggesting a transcriptional regulation activity. The fact that in rodent cells kin17 protein levels increase after gamma-irradiation suggests its participation in a cellular response to ionising radiation. We raised polyclonal antibodies against the whole kin17 protein and against its derived synthetic peptides. We report the detection of kin17 protein and of truncated forms of the protein by Western blot or by immunocytochemistry after transient overexpression in cultured human cells. Our results indicate that the cross-reactivity with the anti-RecA antibodies is due to an antigenic determinant located in the core of kin17 protein, between residues 129 and 228. The kin17 protein is located in the nucleus and is concentrated in small nuclear dot-like structures throughout the nucleoplasm. The RecA homologous region seems to play an essential role in the localisation of kin17 protein since the deletion of this particular region dramatically changes the form and the distribution of the intranuclear foci. We hypothesise that these dot-like structures reflect nuclear metabolism compartmentalization.
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Affiliation(s)
- P Kannouche
- Département de Radiobiologie et de Radiopathologie, Centre d'Etudes de Fontenay-aux-Roses, CEA, France
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34
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Malkov VA, Sastry L, Camerini-Otero RD. RecA protein assisted selection reveals a low fidelity of recognition of homology in a duplex DNA by an oligonucleotide. J Mol Biol 1997; 271:168-77. [PMID: 9268650 DOI: 10.1006/jmbi.1997.1164] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
We have developed an in vitro selection procedure to elucidate the specificity of RecA assisted oligonucleotide recognition of double stranded DNA. The procedure was based on formation of a synaptic complex between an oligonucleotide-RecA filament and a supercoiled plasmid bearing a homologous partially degenerate region. The specificity of the selection depended on the reaction conditions: starting with a population that had, on average, 2.8 randomly distributed mismatches out of 27 bp, a population selected in the presence of 100 mM KCl had on average 1.0 mismatches, while a population selected at low ionic strength was less specific and had, on average, 2.0 mismatches. From the distributions of mismatches observed we calculated that the average destabilization free energy for one mismatch is 1.7(+/-0.5) kcal/mol. This is substantially less than the free energy for the incorporation of one mismatch in naked DNA duplex or a Py-Pu-Py triplex. Thus, RecA has an ability to decrease the fidelity of the homologous pairing reaction and minimize the cost of pairing between similar but not identical sequences. This "antiproofreading" activity of RecA protein does not require ATP hydrolysis.
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Affiliation(s)
- V A Malkov
- Genetics and Biochemistry Branch, National Institutes of Health (NIDDK), Building 10 Room 9D15, 10 Center Drive MSC 1810, Bethesda, MD, 20892-1810, USA
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35
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Baumann P, Benson FE, Hajibagheri N, West SC. Purification of human Rad51 protein by selective spermidine precipitation. Mutat Res 1997; 384:65-72. [PMID: 9298115 DOI: 10.1016/s0921-8777(97)00028-1] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
The human Rad51 protein is a structural homolog of Escherichia coli RecA. The exact role of human Rad51 within the cell is poorly understood but, like its bacterial and yeast homologs, hRad51 is believed to play a central role in homologous recombination. However, recent reports that transgenic mice lacking the RAD51 gene die early in development suggest an additional and essential function for mammalian Rad51 in cell proliferation or genome maintenance. In this paper we describe a simple and quick method for the purification of human Rad51 overproduced in E. coli. Dialysis of cell-free extracts against buffer containing low concentrations of spermidine result in the formation of hRad51 microcrystals as observed by light and electron microscopy. The crystals were easily redissolved in phosphate buffer and hRad51 was further purified to homogeneity using hydroxylapatite, affi-gel heparin and Q-sepharose chromatography. When purified by this method hRad51 is free of endo- and exonuclease activities and suitable for enzymological studies. Spermidine precipitation also provides a rapid method for the large scale purification of hRad51 suitable for physical analysis.
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Affiliation(s)
- P Baumann
- Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, UK
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36
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Himmelreich R, Hilbert H, Plagens H, Pirkl E, Li BC, Herrmann R. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res 1996; 24:4420-49. [PMID: 8948633 PMCID: PMC146264 DOI: 10.1093/nar/24.22.4420] [Citation(s) in RCA: 798] [Impact Index Per Article: 27.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
The entire genome of the bacterium Mycoplasma pneumoniae M129 has been sequenced. It has a size of 816,394 base pairs with an average G+C content of 40.0 mol%. We predict 677 open reading frames (ORFs) and 39 genes coding for various RNA species. Of the predicted ORFs, 75.9% showed significant similarity to genes/proteins of other organisms while only 9.9% did not reveal any significant similarity to gene sequences in databases. This permitted us tentatively to assign a functional classification to a large number of ORFs and to deduce the biochemical and physiological properties of this bacterium. The reduction of the genome size of M. pneumoniae during its reductive evolution from ancestral bacteria can be explained by the loss of complete anabolic (e.g. no amino acid synthesis) and metabolic pathways. Therefore, M. pneumoniae depends in nature on an obligate parasitic lifestyle which requires the provision of exogenous essential metabolites. All the major classes of cellular processes and metabolic pathways are briefly described. For a number of activities/functions present in M. pneumoniae according to experimental evidence, the corresponding genes could not be identified by similarity search. For instance we failed to identify genes/proteins involved in motility, chemotaxis and management of oxidative stress.
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Affiliation(s)
- R Himmelreich
- Zentrum für Molekulare Biologie Heidelberg, Mikrobiologie, Universität Heidelberg, Germany
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37
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Puchta H, Hohn B. From centiMorgans to base pairs: homologous recombination in plants. TRENDS IN PLANT SCIENCE 1996. [PMID: 0 DOI: 10.1016/s1360-1385(96)82595-0] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/16/2023]
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