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1
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Walsh HA. Preterminal protein, the achilles heel of adenoviridae: Implications for adenoviral infections. World J Pharmacol 2024; 13:97723. [DOI: 10.5497/wjp.v13.i2.97723] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/13/2024] [Revised: 10/03/2024] [Accepted: 11/12/2024] [Indexed: 11/29/2024] Open
Abstract
BACKGROUND Adenoviruses pose a serious health risk particularly in the absence of any clinically approved treatment. As adenoviral infections are quite frequent and recent outbreaks manifest more virulent variant strains, the need to develop an effective treatment remains a priority. The adenoviral protein, preterminal protein (pTP), is one of the key common products of the viral lifecycle as it is necessary to initiate viral replication and hence the infection process. This makes pTP a potential chemotherapeutic target in the search for and development of an effective treatment for adenoviral induced infections. Here we report, for the first time, that glycosylation of pTP in situ prevents binding to ssDNA in vitro.
AIM To explore whether specific structural tailoring of the adenoviral protein pTP, imparts the potential to scupper the viral replication process.
METHODS All chemicals used were of reagent grade. Overexpression of pTP was achieved using the ‘BAC to BAC’ expression system. The presence and relative concentration of the protein was determined throughout the incubation period by the Bradford assay. The pTP was identified by MALDI-TOFF and sodium dodecyl sulphate polyacrylamide gel electrophoresis. For the removal of the aminosugar, a deglycosylase enzyme kit from PROZYME was used. Purification of cloned pTP (6xHis) was done with a ssDNA cellulose column followed by a Ni-NTA column. His-tags were excised with the Tobacco etch virus protease. Protein fractionation was performed with a fraction collector coupled to a UV detector (280 nm) from Pharmacia.
RESULTS The pTP overexpressed in insect cells (Spodoptera frugiperda) (> 96 hours), is unable to bind to ssDNA in vitro. Treatment of this unbound protein with a deglycosidase enzyme that is specific for the removal of truncated unsubstituted O-linked Galβ(1-3)GalNAc-α1 disaccharides bound to Thr or Ser in a glycoprotein, restores binding to ssDNA. Data is presented as a linegraph for both the glycosylated and the deglycosylated proteins. Each point represents the mean of triplicate experiments (from different batches). Means and standard deviation were calculated and plotted on a line graph (with error bars).
CONCLUSION The finding that glycosylation of cloned pTP in situ prevents binding to ssDNA in vitro could aid in the development of an effective treatment of adenoviral infections and/or as an adjunct to complement other anti-adenoviral chemotherapeutic strategies.
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Affiliation(s)
- Harold A Walsh
- Faculty of Pharmacy, Division of Pharmacology, Rhodes University, Grahamstown 6139, Eastern Cape, South Africa
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2
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Lamarche BJ, Orazio NI, Goben B, Meisenhelder J, You Z, Weitzman MD, Hunter T. Repair of protein-linked DNA double strand breaks: Using the adenovirus genome as a model substrate in cell-based assays. DNA Repair (Amst) 2018; 74:80-90. [PMID: 30583959 DOI: 10.1016/j.dnarep.2018.12.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2018] [Revised: 12/07/2018] [Accepted: 12/07/2018] [Indexed: 11/29/2022]
Abstract
The DNA double strand breaks (DSBs) created during meiotic recombination and during some types of chemotherapy contain protein covalently attached to their 5' termini. Removal of the end-blocking protein is a prerequisite to DSB processing by non-homologous end-joining or homologous recombination. One mechanism for removing the protein involves CtIP-stimulated Mre11-catalyzed nicking of the protein-linked strand distal to the DSB terminus, releasing the end-blocking protein while it remains covalently attached to an oligonucleotide. Much of what is known about this repair process has recently been deciphered through in vitro reconstitution studies. We present here a novel model system based on adenovirus (Ad), which contains the Ad terminal protein covalently linked to the 5' terminus of its dsDNA genome, for studying the repair of 5' protein-linked DSBs in vivo. It was previously shown that the genome of Ad mutants that lack early region 4 (E4) can be joined into concatemers in vivo, suggesting that the Ad terminal protein had been removed from the genome termini prior to ligation. Here we show that during infection with the E4-deleted Ad mutant dl1004, the Ad terminal protein is removed in a manner that recapitulates removal of end-blocking proteins from cellular DSBs. In addition to displaying a dependence on CtIP, and Mre11 acting as the endonuclease, the protein-linked oligonucleotides that are released from the viral genome are similar in size to the oligos that remain attached to Spo11 and Top2 after they are removed from the 5' termini of DSBs during meiotic recombination and etoposide chemotherapy, respectively. The single nucleotide resolution that is possible with this assay, combined with the single sequence context in which the lesion is presented, make it a useful tool for further refining our mechanistic understanding of how blocking proteins are removed from the 5' termini of DSBs.
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Affiliation(s)
- Brandon J Lamarche
- Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, California, 92037, USA; Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California, 92037, USA
| | - Nicole I Orazio
- Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, California, 92037, USA
| | - Brittany Goben
- Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California, 92037, USA
| | - Jill Meisenhelder
- Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California, 92037, USA
| | - Zhongsheng You
- Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, 63110, USA
| | - Matthew D Weitzman
- Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, California, 92037, USA.
| | - Tony Hunter
- Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California, 92037, USA.
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3
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Pan J, Lin L, Tao YJ. Self-guanylylation of birnavirus VP1 does not require an intact polymerase activity site. Virology 2009; 395:87-96. [PMID: 19801157 DOI: 10.1016/j.virol.2009.09.004] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2009] [Revised: 08/19/2009] [Accepted: 09/04/2009] [Indexed: 11/27/2022]
Abstract
Protein priming is an important mechanism that many viruses use to initiate genomic DNA or RNA synthesis. Birnaviruses are the only double-stranded (ds) RNA viruses that use protein priming. The viral-encoded VP1 of birnavirus functions as both a polymerase and a protein primer and is able to undergo self-guanylylation to acquire a covalently linked rGMP. By employing biochemical assays using recombinant proteins, we have shown that VP1 self-guanylylation does not require an RNA template but is dependent on divalent metal ions. VP1 reacts with all four types of rNTPs but strongly prefers rGTP. Unexpectedly, two fatal polymerase mutants D402A and E421Y, each having an essential catalytic residue mutated and unable to catalyze RNA synthesis, remain active in self-guanylylation. The guanylylation site was further mapped to the VP1 N-terminal domain. Our results support a mechanism in which VP1 self-guanylylation is catalyzed by a novel active site different from the polymerase active site.
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Affiliation(s)
- Junhua Pan
- Department of Biochemistry and Cell Biology, Rice University, 6100 Main Street, Houston, TX 77005, USA
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4
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Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus. Mol Cell Biol 2003. [PMID: 14582191 DOI: 10.1128/mcb.2.12.1492] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60 degrees C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases alpha, beta, gamma and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.
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5
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de Jong RN, van der Vliet PC, Brenkman AB. Adenovirus DNA replication: protein priming, jumping back and the role of the DNA binding protein DBP. Curr Top Microbiol Immunol 2003; 272:187-211. [PMID: 12747551 DOI: 10.1007/978-3-662-05597-7_7] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/11/2023]
Abstract
The adenovirus (Ad) genome is a linear double-stranded (ds) molecule containing about 36 kilobase pairs. At each end of the genome an approximately 100 base pair (bp) inverted terminal repeat (ITR) is found, the exact length depending on the serotype. To the 5'-end of each ITR, a 55-kDa terminal protein (TP) is covalently coupled. The Ad DNA replication system was one of the first replication systems that could be reconstituted in vitro (Challberg and Kelly 1979). The system requires three virally encoded proteins: precursor TP (pTP), DNA polymerase (Pol) and the DNA binding protein (DBP). In addition, three stimulating human cellular proteins have been identified. These are the transcription factors NFI (Nagata et al. 1982) and Oct-1 (Pruijn et al. 1986) and the type I topoisomerase NFII (Nagata et al. 1983). Ad DNA replication uses a protein primer for replication initiation. The transition from initiation to elongation is marked by a jumping back mechanism (King and van der Vliet 1994), followed by elongation. In order to elongate DBP is required. In this review we discuss the roles of DBP during initiation and elongation and we relate biochemical data on the jumping back mechanism used by Ad Pol to the recently solved crystal structure of a Pol alpha-like replication complex (Franklin et al. 2001). We comment on the conditions and possible functions of jumping back and propose a model to describe the jumping back mechanism.
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Affiliation(s)
- R N de Jong
- University Medical Center, Department of Physiological Chemistry and Center for Biomedical Genetics, Utrecht, The Netherlands.
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6
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Brenkman AB, Breure EC, van der Vliet PC. Molecular architecture of adenovirus DNA polymerase and location of the protein primer. J Virol 2002; 76:8200-7. [PMID: 12134025 PMCID: PMC155156 DOI: 10.1128/jvi.76.16.8200-8207.2002] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2002] [Accepted: 05/13/2002] [Indexed: 11/20/2022] Open
Abstract
Adenovirus (Ad) DNA polymerase (pol) belongs to the distinct subclass of the polalpha family of DNA pols that employs the precursor terminal protein (pTP) as primer. Ad pol forms a stable heterodimer with this primer, and together, they bind specifically to the core origin in order to start replication. After initiation of Ad replication, the resulting pTP-trinucleotide intermediate jumps back and pTP starts to dissociate. Compared to free Ad pol, the pTP-pol complex shows reduced polymerase and exonuclease activities, but the reason for this is not understood. Furthermore, the interaction domains between these proteins have not been defined and the contribution of each protein to origin binding is unclear. To address these questions, we used oligonucleotides with a translocation block and show here that pTP binds at the entrance of the primer binding groove of Ad pol, thereby explaining the decreased synthetic activities of the pTP-pol complex and providing insight into how pTP primes Ad replication. Employing an exonuclease-deficient mutant polymerase, we further show that the polymerase and exonuclease active sites of Ad pol are spatially distinct and that the exonuclease activity of Ad pol is located at the N-terminal part of the protein. In addition, by probing the distances between both active sites and the surface of Ad pol, we show that Ad pol binds a DNA region of 14 to 15 nucleotides. Based on these results, a model for binding of the pTP-pol complex at the origin of replication is proposed.
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Affiliation(s)
- Arjan B Brenkman
- University Medical Centre, Department of Physiological Chemistry and Centre for Biomedical Genetics, Utrecht, The Netherlands
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7
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King AJ, Teertstra WR, van der Vliet PC. Dissociation of the protein primer and DNA polymerase after initiation of adenovirus DNA replication. J Biol Chem 1997; 272:24617-23. [PMID: 9305930 DOI: 10.1074/jbc.272.39.24617] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Initiation of adenovirus DNA replication occurs by a jumping back mechanism in which the precursor terminal priming protein (pTP) forms a pTP.trinucleotide complex (pTP.CAT) catalyzed by the viral DNA polymerase (pol). This covalent complex subsequently jumps back 3 bases to permit the start of chain elongation. Before initiation, pTP and pol form a tight heterodimer. We investigated the fate of this pTP.pol complex during the various steps in replication. Employing in vitro initiation and elongation on both natural viral templates and synthetic oligonucleotides followed by glycerol gradient separation of the reaction products, we established that pTP and pol are separated during elongation. Whereas pTP.C and pTP. CA were still bound to the polymerase, after the formation of pTP. CAT 60% of the pTP.pol complex had dissociated. Dissociation coincides with a change in sensitivity to inhibitors and in Km for dNTPs, suggesting a conformational change in the polymerase, both in the active site and in the pTP interaction domain. In agreement with this, the polymerase becomes a more efficient enzyme after release of the pTP primer. We also investigated whether the synthesis of a pTP initiation intermediate is confined to three nucleotides. Employing synthetic oligonucleotide templates with a sequence repeat of two nucleotides (GAGAGAGA ... instead of the natural GTAGTA ... ) we show that G5 rather than G3 is used to start, leading to a pTP. tetranucleotide (CTCT) intermediate that subsequently jumps back. This indicates flexibility in the use of the start site with a preference for the synthesis of three or four nucleotides during initiation rather than two.
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Affiliation(s)
- A J King
- Laboratory for Physiological Chemistry, University of Utrecht, 3508 TA Utrecht, The Netherlands
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8
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Abstract
The delineation of the genetic etiology of cancer makes gene therapy a rational approach for the molecular treatment of cancer. Many gene delivery systems have been developed, with viral vectors being the most effective. Underlying cancer gene therapy protocols is the recognition that quantitative tumor transduction cannot be achieved with the vector systems available at the present time. One way to overcome this problem could be to amplify the transduction efficiency through the use of vectors capable of replicating specifically in tumor cells. We are currently developing an adenoviral vector in which viral replication will be restricted to the target tumor cells by limiting the expression of viral genes essential for the virus replication only to the tumor cells of interest.
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Affiliation(s)
- DT Curiel
- Gene Therapy Program, University of Alabama at Birmingham, 1824 6th Avenue, South, Room 620 Wallace Tumor Institute, Birmingham, AL 35294-3300, USA
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9
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Hay RT, Freeman A, Leith I, Monaghan A, Webster A. Molecular interactions during adenovirus DNA replication. Curr Top Microbiol Immunol 1995; 199 ( Pt 2):31-48. [PMID: 7555069 DOI: 10.1007/978-3-642-79499-5_2] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Affiliation(s)
- R T Hay
- School of Biological and Medical Sciences, University of St. Andrews, Scotland, UK
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10
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Paul AV, Cao X, Harris KS, Lama J, Wimmer E. Studies with poliovirus polymerase 3Dpol. Stimulation of poly(U) synthesis in vitro by purified poliovirus protein 3AB. J Biol Chem 1994. [DOI: 10.1016/s0021-9258(19)62027-2] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
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11
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Abstract
Using a reconstituted system for adenovirus DNA replication we tested the requirements for ATP and divalent cations. At the standard Mg2+ concentration ATP stimulated initiation 5 to 10-fold. However, this effect was caused by complexing Mg2+. At the optimal Mg2+ concentration ATP was not required for initiation or elongation. Besides Mg2+ also Mn2+, Ca2+ and Ba2+ were shown to support initiation whereas for elongation only Mg2+ was accepted. Since Mn2+ could efficiently be used for DNA chain elongation on synthetic templates we hypothesize that Mg2+ is essential for the transition of initiation to elongation.
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Affiliation(s)
- R Pronk
- Laboratory for Physiological Chemistry, Utrecht University, The Netherlands
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12
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Zoulim F, Seeger C. Reverse transcription in hepatitis B viruses is primed by a tyrosine residue of the polymerase. J Virol 1994; 68:6-13. [PMID: 7504742 PMCID: PMC236258 DOI: 10.1128/jvi.68.1.6-13.1994] [Citation(s) in RCA: 196] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
All known DNA polymerases require primers for the initiation of DNA synthesis. While cellular polymerases and reverse transcriptases use free hydroxyl groups of RNA or DNA, the DNA polymerases of certain animal viruses and bacteriophages depend upon hydroxyl groups of amino acid residues within proteins as primers for DNA synthesis. Recently, the reverse transcriptase of a hepadnavirus has been shown to prime RNA-directed DNA synthesis from an internal site of the polypeptide (G.H. Wang and C. Seeger, Cell 71:663-670, 1992). In this report we demonstrate that a tyrosine residue of the polymerase polypeptide is the site of a phosphodiester linkage with the first nucleotide of minus-strand DNA. This tyrosine residue is located within an amino-terminal domain of the polymerase polypeptide and is indispensable for the priming of reverse transcription. Our results demonstrate that the hepatitis B virus reverse transcriptase can initiate DNA synthesis without the requirement for tRNA as a primer.
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Affiliation(s)
- F Zoulim
- Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
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13
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Caldentey J, Blanco L, Savilahti H, Bamford DH, Salas M. In vitro replication of bacteriophage PRD1 DNA. Metal activation of protein-primed initiation and DNA elongation. Nucleic Acids Res 1992; 20:3971-6. [PMID: 1324473 PMCID: PMC334074 DOI: 10.1093/nar/20.15.3971] [Citation(s) in RCA: 31] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Compared to Mg2+, the use of Mn2+ as the metal activator of the phage DNA polymerase results in a great stimulation of the initiation reaction. The molecular basis of the observed stimulatory effect is an increase in the velocity of the reaction. The phage DNA polymerase is also able to catalyze the formation of the initiation complex in the absence of DNA template. Although the presence of Mn2+ does not affect either the polymerization activity or the processivity of the DNA polymerase, this metal is unable to activate the overall replication of the phage genome. This can be explained by a deleterious effect of Mn2+ on the 3'-5'-exonucleolytic and/or the strand-displacement activity, the latter being an intrinsic function of the viral DNA polymerase required for protein-primed DNA replication.
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Affiliation(s)
- J Caldentey
- Department of Genetics, University of Helsinki, Finland
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14
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Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex. J Biol Chem 1990. [DOI: 10.1016/s0021-9258(17)44799-5] [Citation(s) in RCA: 51] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
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15
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Watson CJ, Hay RT. Expression of adenovirus type 2 DNA polymerase in insect cells infected with a recombinant baculovirus. Nucleic Acids Res 1990; 18:1167-73. [PMID: 2181402 PMCID: PMC330431 DOI: 10.1093/nar/18.5.1167] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Sequences encoding adenovirus type 2 DNA polymerase were placed under control of the polyhedrin promoter and inserted into the baculovirus Autographa californica nuclear polyhedrosis virus by homologous recombination. Insect cells infected with the recombinant virus produced substantial amounts of the adenovirus type 2 DNA polymerase protein which was functional in both DNA polymerase and replication initiation reactions. Thus, the baculovirus expression system can provide active adenovirus type 2 DNA polymerase that is produced in quantities suitable for biochemical and structural analysis.
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MESH Headings
- Adenoviruses, Human/enzymology
- Adenoviruses, Human/genetics
- Animals
- Base Sequence
- Cell Fractionation
- Cells, Cultured
- Cloning, Molecular
- DNA Polymerase II/biosynthesis
- DNA Polymerase II/genetics
- DNA Polymerase II/metabolism
- DNA Replication
- DNA, Recombinant/genetics
- DNA, Recombinant/metabolism
- DNA, Viral/genetics
- DNA, Viral/metabolism
- Gene Expression Regulation, Viral
- Insect Viruses/genetics
- Molecular Sequence Data
- Moths
- Promoter Regions, Genetic
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Affiliation(s)
- C J Watson
- Department of Biochemistry and Microbiology, University of St Andrews, Fife, UK
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16
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Pettit SC, Horwitz MS, Engler JA. Mutations of the precursor to the terminal protein of adenovirus serotypes 2 and 5. J Virol 1989; 63:5244-50. [PMID: 2511338 PMCID: PMC251189 DOI: 10.1128/jvi.63.12.5244-5250.1989] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Using a series of transient expression plasmids and adenovirus-specific DNA replication assays for both initiation and elongation, we measured the relative activities of mutant polypeptides of the precursor to the terminal protein (pTP) in vitro. Mutations that removed two to six amino acids of the amino terminus gradually decreased pTP activity; a deletion of 18 amino acids was completely inactive. Replacement of cysteine at residue 8 with a serine had little effect on pTP activity. Two amino-terminal in-frame linker insertion mutant polypeptides previously characterized in vivo as either replication defective or temperature sensitive had considerable activity at the permissive temperature in vitro. For one mutant pTP with a temperature-sensitive phenotype in vivo, elongation activity was decreased more than initiation in vitro, suggesting a role for this protein after the initiation step. Replacement mutations of serine 580, the site of covalent attachment of dCTP, completely abolished pTP function for both initiation and elongation.
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Affiliation(s)
- S C Pettit
- Department of Biochemistry, University of Alabama, Birmingham, University Station 35294
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17
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Chen M, Horwitz MS. Dissection of functional domains of adenovirus DNA polymerase by linker-insertion mutagenesis. Proc Natl Acad Sci U S A 1989; 86:6116-20. [PMID: 2548198 PMCID: PMC297786 DOI: 10.1073/pnas.86.16.6116] [Citation(s) in RCA: 29] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Linker-insertion mutations were introduced into the cloned adenovirus DNA polymerase gene and the functional effects on the initiation and elongation of DNA in vitro were measured. Essential regions of the polymerase appear to be scattered in patches across the entire molecule and are not limited to the five regions of homology shared with a variety of other replicating polymerases. Thus, the adenovirus DNA polymerase presumably contains active sites that must be formed by distant interactions across the polymerase molecule.
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Affiliation(s)
- M Chen
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461
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18
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Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules. Mol Cell Biol 1989. [PMID: 2540421 DOI: 10.1128/mcb.9.2.541] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.
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19
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Porter SE, Champoux JJ. Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules. Mol Cell Biol 1989; 9:541-50. [PMID: 2540421 PMCID: PMC362630 DOI: 10.1128/mcb.9.2.541-550.1989] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.
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Affiliation(s)
- S E Porter
- Department of Microbiology, School of Medicine, University of Washington, Seattle 98195
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20
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21
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Goetz GS, Hurwitz J. Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. I. Replication of DNA containing an alteration in position 1 of the 30-nucleotide icosahedral bacteriophage origin. J Biol Chem 1988. [DOI: 10.1016/s0021-9258(18)37610-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022] Open
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22
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Young C, Nester EW. Association of the virD2 protein with the 5' end of T strands in Agrobacterium tumefaciens. J Bacteriol 1988; 170:3367-74. [PMID: 3403506 PMCID: PMC211303 DOI: 10.1128/jb.170.8.3367-3374.1988] [Citation(s) in RCA: 117] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
The soil bacterium Agrobacterium tumefaciens can incite tumors in many dicotyledonous plants by transferring a portion (T-DNA) of its Ti plasmid into susceptible plant cells. The T-DNA is flanked by border sequences that serve as recognition sites for specific cleavage by an endonuclease that comprises two virD-encoded proteins (VirD1 and VirD2). After cleavage, both double-stranded, nicked T-DNA molecules and single-stranded T-DNA molecules (T strands) were present. We have determined that a protein is tightly associated with, and probably covalently attached to, the 5' end of the T strands. Analysis of deletion derivatives in Escherichia coli, immunoprecipitation, and a procedure combining immunoblot and nucleic acid hybridization data identified this protein as the gene product of virD2.
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Affiliation(s)
- C Young
- Department of Microbiology, University of Washington, Seattle 98195
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23
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Abstract
By manipulating an infectious cDNA clone of poliovirus, we have introduced a single-codon insertion into the 3A region of the viral genome which has been proposed to encode a functional precursor of the virion-linked protein VPg. The resulting mutant was cold sensitive in monkey kidney cells. Viral RNA synthesis was poor at 32.5 degrees C, although no other function of the virus was obviously affected. The synthesis of both positive and negative strands was severely depressed. Temperature shift experiments suggest that a normal level of production of the affected function was required only during the early (exponential) phase of RNA synthesis. Analysis of viral polyprotein processing at the nonpermissive temperature revealed that some of the normal cleavages were not made, most likely as a consequence of the defect in RNA synthesis or as a result of the concomitant reduction in the level of virally encoded proteases.
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Affiliation(s)
- H D Berstein
- Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142
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24
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Initiation of adenovirus DNA replication. I. Mechanism of action of a host protein required for replication of adenovirus DNA templates devoid of the terminal protein. J Biol Chem 1988. [DOI: 10.1016/s0021-9258(19)81589-2] [Citation(s) in RCA: 30] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
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25
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Kenny MK, Hurwitz J. Initiation of adenovirus DNA replication. II. Structural requirements using synthetic oligonucleotide adenovirus templates. J Biol Chem 1988. [DOI: 10.1016/s0021-9258(19)81590-9] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
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26
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Remenick J, Kenny MK, McGowan JJ. Inhibition of adenovirus DNA replication by vesicular stomatitis virus leader RNA. J Virol 1988; 62:1286-92. [PMID: 2831388 PMCID: PMC253139 DOI: 10.1128/jvi.62.4.1286-1292.1988] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Vesicular stomatitis virus (VSV) leader RNA and a synthetic oligodeoxynucleotide of the same sequence were found to inhibit the replication of adenovirus DNA in vitro. In contrast, the small RNA transcribed by the VSV defective interfering particle DI-011 did not prevent adenovirus DNA replication. The inhibition produced by leader RNA was at the level of preterminal protein (pTP)-dCMP complex formation, the initiation step of adenovirus DNA replication. Initiation requires the adenovirus pTP-adenovirus DNA polymerase complex (pTP-Adpol), the adenovirus DNA-binding protein, and nuclear factor I. Specific replication in the presence of leader RNA was restored when the concentration of adenovirus-infected or uninfected nuclear extract was increased or by the addition of purified pTP-Adpol or HeLa cell DNA polymerase alpha-primase to inhibited replication reactions. Furthermore, the activities of both purified DNA polymerases could be inhibited by the leader sequence. These results suggest that VSV leader RNA is the viral agent responsible for inhibition of adenovirus and possibly cellular DNA replication during VSV infection.
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Affiliation(s)
- J Remenick
- Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814
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27
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Stunnenberg HG, Lange H, Philipson L, van Miltenburg RT, van der Vliet PC. High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus. Nucleic Acids Res 1988; 16:2431-44. [PMID: 3362670 PMCID: PMC336381 DOI: 10.1093/nar/16.6.2431] [Citation(s) in RCA: 88] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system.
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28
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Pettit SC, Horwitz MS, Engler JA. Adenovirus preterminal protein synthesized in COS cells from cloned DNA is active in DNA replication in vitro. J Virol 1988; 62:496-500. [PMID: 3336069 PMCID: PMC250560 DOI: 10.1128/jvi.62.2.496-500.1988] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Replication of the DNA genome of human adenovirus serotype 2 requires three virus-encoded proteins. Two of these proteins, the preterminal protein (pTP) and the adenovirus DNA polymerase, are transcribed from a single promoter at early times after virus infection. The mRNAs for these proteins share several exons, including one encoded near adenovirus genome coordinate 39. By using plasmids containing DNA fragments postulated to encode the various exons of pTP mRNA, the contributions of each exon to the synthesis of an active pTP have been measured. Only plasmids that contain both the open reading frame for pTP (genome coordinates 29.4 to 23.9) and the HindIII J fragment that contains the exon at genome coordinate 39 can express functional pTP.
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Affiliation(s)
- S C Pettit
- Department of Biochemistry, University of Alabama, Birmingham 35294
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29
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O'Neill EA, Kelly TJ. Purification and characterization of nuclear factor III (origin recognition protein C), a sequence-specific DNA binding protein required for efficient initiation of adenovirus DNA replication. J Biol Chem 1988. [DOI: 10.1016/s0021-9258(19)35442-0] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
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30
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Flomenberg PR, Chen M, Horwitz MS. Characterization of a major histocompatibility complex class I antigen-binding glycoprotein from adenovirus type 35, a type associated with immunocompromised hosts. J Virol 1987; 61:3665-71. [PMID: 2960830 PMCID: PMC255977 DOI: 10.1128/jvi.61.12.3665-3671.1987] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Adenovirus type 35 (Ad35) is a group B adenovirus that has been isolated primarily from patients with acquired immunodeficiency syndrome and other immunodeficiency disorders. We have studied the interaction of this unique adenovirus with the immune system by analyzing Ad35 early viral proteins in infected HeLa cells. We have identified a 29,000-Mr Ad35 early glycoprotein, E29, which associates with class I antigens of the major histocompatibility complex (MHC) in the endoplasmic reticulum. Ad35 E29 is analogous to the group C Ad2 early glycoprotein E3-19K (E19), which has been shown to interfere with the expression of class I antigens on the cell surface (H. Burgert and S. Kvist, Cell 41:987-997, 1985). In contrast to the Ad2 glycoprotein, Ad35 E29 was synthesized in much smaller amounts, was more extensively glycosylated, and did not cross-react with polyclonal antibody against the Ad2 protein. As a control, a class I antigen-binding glycoprotein from another group B adenovirus, Ad7, was also characterized and was found to have properties similar to those of Ad35 E29. Therefore, the differences in the glycosylation and quantity of class I antigen-binding glycoproteins between Ad35 and Ad2 are group related. Inhibition of the expression of MHC class I antigens, which are needed for cytotoxic-T-lymphocyte recognition of virus-infected cells, appears to play a vital role in the adenovirus life cycle in vivo. Our data indicate that this function has been conserved despite significant differences in the MHC class I antigen-binding glycoprotein and in the pathogenicity between serotypes.
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Affiliation(s)
- P R Flomenberg
- Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461
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31
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Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication. Mol Cell Biol 1987. [PMID: 3821731 DOI: 10.1128/mcb.7.2.875] [Citation(s) in RCA: 72] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.
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32
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Adenovirus origin of DNA replication: sequence requirements for replication in vitro. Mol Cell Biol 1987. [PMID: 3821730 DOI: 10.1128/mcb.7.2.864] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions.
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33
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Wides RJ, Challberg MD, Rawlins DR, Kelly TJ. Adenovirus origin of DNA replication: sequence requirements for replication in vitro. Mol Cell Biol 1987; 7:864-74. [PMID: 3821730 PMCID: PMC365145 DOI: 10.1128/mcb.7.2.864-874.1987] [Citation(s) in RCA: 33] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions.
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34
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Rosenfeld PJ, O'Neill EA, Wides RJ, Kelly TJ. Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication. Mol Cell Biol 1987; 7:875-86. [PMID: 3821731 PMCID: PMC365146 DOI: 10.1128/mcb.7.2.875-886.1987] [Citation(s) in RCA: 78] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.
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35
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Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay. Mol Cell Biol 1987. [PMID: 3785168 DOI: 10.1128/mcb.6.5.1363] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol of nuclear factor I-binding site.
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36
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Vartapetian AB, Bogdanov AA. Proteins covalently linked to viral genomes. PROGRESS IN NUCLEIC ACID RESEARCH AND MOLECULAR BIOLOGY 1987; 34:209-51. [PMID: 3326040 DOI: 10.1016/s0079-6603(08)60497-3] [Citation(s) in RCA: 27] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
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37
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Abstract
The adenovirus (Ad) DNA origin of replication was defined through an analysis of the DNA sequences necessary for the replication of plasmid DNAs with purified viral and cellular proteins. Results from several laboratories have shown that the origin consists of two functionally distinct domains: a 10-base-pair sequence present in the inverted terminal repetition (ITR) of all human serotypes and an adjacent sequence constituting the binding site for a cellular protein, nuclear factor I. To determine whether the same nucleotide sequences are necessary for origin function in vivo, we developed an assay for the replication of plasmid DNAs transfected into Ad5-infected cells. The assay is similar to that described by Hay et al. (J. Mol. Biol. 175:493-510, 1984). With this assay, plasmid DNA replication is dependent upon prior infection of cells with virus and only occurs with linear DNA molecules containing viral terminal sequences at each end. Replicated DNA is resistant to digestion with lambda-exonuclease, suggesting that a protein is covalently bound at both termini. A plasmid containing only the first 67 base pairs of the Ad2 ITR replicates as well as plasmids containing the entire ITR. Deletions or point mutations which reduce the binding of nuclear factor I to DNA in vitro reduce the efficiency of plasmid replication in vivo. A point mutation within the 10-base-pair conserved sequence has a similar effect upon replication. These results suggest that the two sequence domains of the Ad origin identified by in vitro studies are in fact important for viral DNA replication in infected cells. In addition, we found that two separate point mutations which lie outside these two sequence domains, and which have little or no effect upon DNA replication in vitro, also reduce the apparent efficiency of plasmid replication in vivo. Thus, there may be elements of the Ad DNA origin of replication which have not yet been identified by in vitro studies.
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38
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Gronostajski RM. Analysis of nuclear factor I binding to DNA using degenerate oligonucleotides. Nucleic Acids Res 1986; 14:9117-32. [PMID: 3786147 PMCID: PMC311933 DOI: 10.1093/nar/14.22.9117] [Citation(s) in RCA: 89] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Nuclear factor I (NFI) binds tightly to DNA containing the consensus sequence TGG(N)6-7GCCAA. To study the role of the spacing between the TGG and GCCAA motifs, oligonucleotides homologous to the NFI binding site FIB-2 were synthesized and used for binding assays in vitro. The wild-type site (FIB-2.6) has a 6bp spacer region and binds tightly to NFI. When the size of this spacer was altered by +/- 1 or 2bp the binding to NFI was abolished. To further assess the role of the spacer and bases flanking the motifs, two oligonucleotide libraries were synthesized. Each member of these libraries had intact TGG and GCCAA motifs, but the sequence of the spacer and the 3bp next to each motif was degenerate. The library with a 6bp spacer bound to NFI to 40-50% the level of FIB-2.6. The library with a 7bp spacer bound to NFI to only 4% the level of FIB-2.6 and some of this binding was weaker than that of FIB-2.6 DNA. This novel use of degenerate DNA libraries has shown that: 1) the structural requirements for FIB sites with a 7bp spacer are more stringent than for sites with a 6bp spacer and 2) a limited number of DNA structural features can prevent the binding of NFI to sites with intact motifs and a 6bp spacer region.
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39
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Lindenbaum JO, Field J, Hurwitz J. The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates. J Biol Chem 1986. [DOI: 10.1016/s0021-9258(18)67513-1] [Citation(s) in RCA: 27] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022] Open
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40
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Bernstein JA, Porter JM, Challberg MD. Template requirements for in vivo replication of adenovirus DNA. Mol Cell Biol 1986; 6:2115-24. [PMID: 3785188 PMCID: PMC367752 DOI: 10.1128/mcb.6.6.2115-2124.1986] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
The adenovirus (Ad) DNA origin of replication was defined through an analysis of the DNA sequences necessary for the replication of plasmid DNAs with purified viral and cellular proteins. Results from several laboratories have shown that the origin consists of two functionally distinct domains: a 10-base-pair sequence present in the inverted terminal repetition (ITR) of all human serotypes and an adjacent sequence constituting the binding site for a cellular protein, nuclear factor I. To determine whether the same nucleotide sequences are necessary for origin function in vivo, we developed an assay for the replication of plasmid DNAs transfected into Ad5-infected cells. The assay is similar to that described by Hay et al. (J. Mol. Biol. 175:493-510, 1984). With this assay, plasmid DNA replication is dependent upon prior infection of cells with virus and only occurs with linear DNA molecules containing viral terminal sequences at each end. Replicated DNA is resistant to digestion with lambda-exonuclease, suggesting that a protein is covalently bound at both termini. A plasmid containing only the first 67 base pairs of the Ad2 ITR replicates as well as plasmids containing the entire ITR. Deletions or point mutations which reduce the binding of nuclear factor I to DNA in vitro reduce the efficiency of plasmid replication in vivo. A point mutation within the 10-base-pair conserved sequence has a similar effect upon replication. These results suggest that the two sequence domains of the Ad origin identified by in vitro studies are in fact important for viral DNA replication in infected cells. In addition, we found that two separate point mutations which lie outside these two sequence domains, and which have little or no effect upon DNA replication in vitro, also reduce the apparent efficiency of plasmid replication in vivo. Thus, there may be elements of the Ad DNA origin of replication which have not yet been identified by in vitro studies.
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41
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Diffley JF, Stillman B. Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay. Mol Cell Biol 1986; 6:1363-73. [PMID: 3785168 PMCID: PMC367659 DOI: 10.1128/mcb.6.5.1363-1373.1986] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol of nuclear factor I-binding site.
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42
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Reconstruction of adenovirus replication origins with a human nuclear factor I binding site. J Biol Chem 1986. [DOI: 10.1016/s0021-9258(17)35788-5] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
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43
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Prelich G, Stillman BW. Functional characterization of thermolabile DNA-binding proteins that affect adenovirus DNA replication. J Virol 1986; 57:883-92. [PMID: 2936900 PMCID: PMC252818 DOI: 10.1128/jvi.57.3.883-892.1986] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
The human adenovirus type 2 (Ad2) mutant Ad2ts111 has previously been shown to contain two mutations which result in a complex phenotype. Ad2ts111 contains a single base change in the early region 1B (E1B) 19,000-molecular-weight (19K) coding region which yields a cyt deg phenotype and another defect which maps to the E2A 72K DNA-binding protein (DBP) coding region that causes a temperature-sensitive DNA replication phenotype. Here we report that the defect in the Ad2ts111 DBP is due to a single G----T transversion that results in a substitution of valine for glycine at amino acid 280. A temperature-independent revertant, Ad2ts111R10, was isolated, which reverts back to glycine at amino acid 280 yet retains the cyt and deg phenotypes caused by the 19K mutation. We physically separated the two mutations of Ad2ts111 by constructing a recombinant virus, Ad2ts111A, which contained a wild-type Ad2 E1B 19K gene and the gly----val mutation in the 72K gene. Ad2ts111A was cyt+ deg+, yet it was still defective for DNA replication at the nonpermissive temperature. The Ad2ts111 DBP mutation is located only two amino acids away from the site of the mutation in Ad2+ND1ts23, a previously sequenced DBP mutant. Biochemical studies of purified Ad2+ND1ts23 DBP showed that this protein was defective for elongation but not initiation of replication in a cell-free replication system consisting of purified Ad polymerase, terminal protein precursor, and nuclear factor I. Ad2+ND1ts23 DBP bound less tightly to single-strand DNA than did Ad2 DBP, as shown by salt gradient elution of purified DBPs from denatured DNA cellulose columns. This decreased binding to DNA was probably due to local conformational changes in the protein at a site that is critical for DNA binding rather than to global changes in protein structure, since both the Ad2+ND1ts23 and Ad2 DBPs showed identical cleavage patterns by the protease thermolysin at various temperatures.
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44
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Rosenfeld PJ, Kelly TJ. Purification of nuclear factor I by DNA recognition site affinity chromatography. J Biol Chem 1986. [DOI: 10.1016/s0021-9258(17)36106-9] [Citation(s) in RCA: 170] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
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Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites. Mol Cell Biol 1985. [PMID: 4039788 DOI: 10.1128/mcb.5.5.964] [Citation(s) in RCA: 71] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.
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de Vries E, van Driel W, Tromp M, van Boom J, van der Vliet PC. Adenovirus DNA replication in vitro: site-directed mutagenesis of the nuclear factor I binding site of the Ad2 origin. Nucleic Acids Res 1985; 13:4935-52. [PMID: 4040630 PMCID: PMC321836 DOI: 10.1093/nar/13.13.4935] [Citation(s) in RCA: 75] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The template requirements for efficient adenovirus DNA replication were studied in vitro in a reconstituted system with cloned DNA fragments, containing the Ad2 origin region, as templates. Replication is enhanced by nuclear factor I, a cellular protein that binds specifically to the Ad2 origin. This stimulation is shown to be strongly dependent on the concentration of the adenovirus DNA binding protein. Using synthetic oligonucleotides we have constructed plasmids with base substitutions in the nuclear factor I binding region. Footprint analysis and competition filter binding studies show that two of the three small blocks of conserved nucleotides in this region are involved in the binding of nuclear factor I. The binding affinity can be influenced by the base composition of the degenerate region just outside these two blocks. In vitro initiation and DNA chain elongation experiments with the mutants demonstrate that binding of nuclear factor I to the Ad2 origin is necessary for stimulation. However, binding alone is not always sufficient since a mutation which only slightly disturbs binding is strongly impaired in stimulation of DNA replication by nuclear factor I.
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Friefeld BR, Korn R, de Jong PJ, Sninsky JJ, Horwitz MS. The 140-kDa adenovirus DNA polymerase is recognized by antibodies to Escherichia coli-synthesized determinants predicted from an open reading frame on the adenovirus genome. Proc Natl Acad Sci U S A 1985; 82:2652-6. [PMID: 2581253 PMCID: PMC397622 DOI: 10.1073/pnas.82.9.2652] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Sequence studies of the adenovirus 2 genome have revealed the presence of a large open reading frame (ORF) from 22.9 to 14.2 map units that is believed to encode most of the adenovirus DNA polymerase (Ad Pol). An 838-base-pair fragment (19.6-17.3 map units) containing approximately 25% of this ORF has been cloned and expressed in a beta-galactosidase-chloramphenicol acetyltransferase (lacZ-CAT) expression vector under the control of the trp-lac hybrid promoter. This recombinant vector directed the synthesis of a 58-kDa lacZ-Ad Pol-CAT fusion protein that has CAT activity. This fusion protein was easily purified by affinity chromatography in which chloramphenicol, the substrate for CAT, was covalently bound to a matrix. Antisera were prepared against the purified 58-kDa lacZ-Ad Pol-CAT fusion protein and were found to react specifically with the 140-kDa Ad Pol by ELISA and immunoblot analysis. In addition, these antisera recognized 120- and 29-kDa polypeptides in immunoblot analysis of partially purified terminal protein precursor (pTP)-Ad Pol complex. The exact nature of the 120- and 29-kDa polypeptides is not known, but they may be breakdown products of Ad Pol. Although the lacZ-Ad Pol-CAT fusion protein is not active in any of the Ad Pol enzymatic reactions, antibody against the prokaryotic fusion protein should be useful for screening bacteria harboring plasmids that have been constructed to express the entire Ad Pol ORF.
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Gronostajski RM, Adhya S, Nagata K, Guggenheimer RA, Hurwitz J. Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites. Mol Cell Biol 1985; 5:964-71. [PMID: 4039788 PMCID: PMC366811 DOI: 10.1128/mcb.5.5.964-971.1985] [Citation(s) in RCA: 66] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.
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Watabe K, Leusch M, Ito J. Replication of bacteriophage phi 29 DNA in vitro: the roles of terminal protein and DNA polymerase. Proc Natl Acad Sci U S A 1984; 81:5374-8. [PMID: 6433349 PMCID: PMC391706 DOI: 10.1073/pnas.81.17.5374] [Citation(s) in RCA: 46] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
phi 29 DNA replication is initiated by the formation of a covalent complex between the viral-coded terminal protein and dAMP (TP-dAMP). This initiation reaction system has been reconstituted from two phage-encoded proteins, the terminal protein and DNA polymerase. The phi 29 DNA polymerase was purified from phage-infected cells by using poly(dA) X p(dT)12-18 as an assay template. The purified polymerase has an apparent molecular mass of 68 kDa in its native form and it appears to function as a monomer. The terminal protein was purified to homogeneity from Escherichia coli cells harboring a cloned plasmid that contained a phi 29 gene 3 segment. The molecular mass of the purified terminal protein was about 30 kDa in both the denatured and the native form. The protein apparently functions as a monomer. When the terminal protein and DNA polymerase were incubated in the presence of dATP, Mg2+, and phi 29 DNA-protein as template, the terminal protein bound covalently to dAMP. This reaction did not require ATP. In addition, these two purified fractions catalyzed DNA chain elongation from both ends of phi 29 DNA, yielding the expected 9- to 12-base fragment when assayed in the presence of 2',3'-dideoxycytidine triphosphate. These results indicate that phi 29 DNA polymerase catalyzes formation of the terminal protein-dAMP complex and can also catalyze chain elongation at least 9-12 bases from both ends of phi 29 DNA.
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Matsumoto K, Saito T, Kim CI, Ando T, Hirokawa H. Bacteriophage phi 29 DNA replication in vitro: participation of the terminal protein and the gene 2 product in elongation. MOLECULAR & GENERAL GENETICS : MGG 1984; 196:381-6. [PMID: 6438445 DOI: 10.1007/bf00436183] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
From phi 29-infected Bacillus subtilis cells, we have isolated a protein fraction which promotes in vitro replication of phi 29 DNA. This fraction catalyses both initiation and elongation, indicating that it contains the product of gene 3 (tp: terminal protein) and the product of gene 2 (gp2: probably a DNA polymerase), since initiation requires the two products (Blanco et al. 1983; Matsumoto et al. 1983). The fractions isolated from cells infected with temperature-sensitive (ts) mutants of gene 2 and gene 3 were thermolabile in both the initiation and elongation assays. When the pre-initiated material from the ts fractions of each mutant was heat-inactivated and mixed no complementation, restoring the elongation activity, was found. These results indicate: (i) tp and gp2 participate not only in the initiation but also in the elongation of phi 29 DNA replication, (ii) they probably function in tight physical association with each other.
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