Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Extracts of insect cells infected with baculoviruses recombinant for the herpes simplex virus 1 (HSV-1)-encoded enzymes that are required for its replication can promote the rolling circle replication of circular plasmid templates. Replication is independent of a HSV-1 origin of replication (oris) or the HSV-1 origin binding protein and is inhibited by the origin binding protein when the plasmid contains oris. Replication is dependent on a complex composed of the HSV-1-encoded DNA polymerase and its processivity enhancing factor (the UL42 protein), ICP8 (the HSV-1-encoded single-strand DNA binding protein), and the HSV-1-encoded helicase-primase. The complex can be purified by size-exclusion and anion-exchange chromatography.
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PMID:Rolling circle DNA replication in vitro by a complex of herpes simplex virus type 1-encoded enzymes. 793 10

Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination.
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PMID:Biochemistry of homologous recombination in Escherichia coli. 796 21

By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa californica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2, and PE38 stimulate DNA replication. No stimulation by the AcMNPV pcna gene, encoding a protein with sequence homology to proliferating-cell nuclear antigen, was observed. A pattern of amino acids found in a number of single-stranded-DNA-binding proteins was identified in the carboxyl-terminal region of IE-1.
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PMID:Identification of genes involved in DNA replication of the Autographa californica baculovirus. 797 36

Persistent infection by papillomaviruses involves the maintenance of viral DNA as a nuclear plasmid, the replication of which requires host DNA polymerases. The role of the cellular DNA polymerase alpha-primase holoenzyme was probed by using soluble extracts from rodent cells that replicate bovine papilloma virus 1 and human papilloma virus 6b DNA in the presence of the viral E1 helicase and the E2 transcription factor. Monoclonal antibodies directed against the catalytic 180-kDa subunit of polymerase alpha inhibit DNA synthesis in this system. Addition of purified human polymerase alpha-primase holoenzyme to neutralized extracts restores their DNA synthetic activity. The amino-terminal 424 amino acids of E1 forms a specific protein complex with the p180 polymerase subunit. Immune complexes can be isolated with antibodies directed against E1 that contain a DNA polymerase activity. Moreover, this polymerase activity can be neutralized by anti-polymerase alpha antibodies. Permissivity barriers were not encountered in this in vitro system, as bovine E1 can interface with the murine and human replication apparatus. Although the large tumor antigens encoded by simian virus 40 and polyoma share limited primary sequence homology with the papillomavirus E1 proteins, the organization of functional motifs at the level of primary protein structure is remarkably similar. In addition to their origin-specific DNA-binding activity, each of these helicases may function to help recruit the cellular polymerase alpha-primase complex to the viral replication origin.
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PMID:The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the viral E1 helicase. 807 45

The herpes simplex virus type 1 (HSV) UL5, UL8, and UL52 proteins form a helicase-primase complex in infected cells. Several laboratories have demonstrated that helicase and nucleoside triphosphatase activities of the heterotrimer (UL5/8/52) are indistinguishable from that of a subassembly of UL5 and UL52 (UL5/52). Although the UL5/52 subassembly functions in coupled primase-polymerase assays on homopolymeric templates, its activity on natural DNA templates has been reported to require UL8. To determine the role of UL8 in primase assays, the activity of the UL5/52 subassembly was compared to that of the heterotrimer reconstituted by adding UL8 to UL5/52. We detected significant activity of the UL5/52 subassembly in coupled primase-polymerase and oligoribonucleotide primer synthesis assays on phi X174 and M13 virion DNAs. However the addition of UL8 to UL5/52 stimulated this activity in a dose-dependent manner. We demonstrate that stimulation occurred at the level of primer synthesis. UL8 did not affect the amount or size of primers annealed to template, their utilization by DNA polymerase, or the use of specific initiation sites within the template. In kinetic studies, the rate of primer synthesis was increased by UL8 but the Km for phi X174 DNA template was unchanged. These results suggest that a function of the UL8 component of the HSV helicase-primase complex is to increase the efficiency of primer synthesis by UL5/52.
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PMID:The UL8 component of the herpes simplex virus helicase-primase complex stimulates primer synthesis by a subassembly of the UL5 and UL52 components. 810 78

The product of gene 2.5 protein of bacteriophage T7, a single-stranded DNA-binding protein, physically interacts with phage encoded DNA polymerase and primase/helicase proteins. A truncated gene 2.5 protein (GP2.5-delta 21C) was constructed by in vitro mutagenesis and lacks the 21 carboxyl-terminal amino acids found in wild-type gene 2.5 protein, 15 of which are acidic. GP2.5-delta 21C cannot substitute for wild-type gene 2.5 protein in vivo; the phage are not viable and exhibit less than 1% of the DNA synthesis observed in wild-type phage-infected cells. GP2.5-delta 21C has been purified to apparent homogeneity from cells overexpressing its cloned gene and has a conformation that differs from that of the wild-type gene 2.5 protein as judged by its circular dichroism spectra. Purified GP2.5-delta 21C retains its ability to bind to single-stranded DNA; the association constant of the protein for single-stranded DNA, determined by nitrocellulose filter binding, is 3.2 x 10(6) M-1 and is identical to that determined for wild-type gene 2.5 protein. However, GP2.5-delta 21C is a monomer in solution, whereas the wild-type protein exists as a dimer. GP2.5-delta 21C does not physically interact with T7 DNA polymerase as measured by affinity chromatography and fluorescent emission anisotropy. The mutant protein cannot stimulate T7 DNA polymerase activity on primed single-stranded DNA templates.
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PMID:Acidic carboxyl-terminal domain of gene 2.5 protein of bacteriophage T7 is essential for protein-protein interactions. 810 11

The p107 protein is related to the Rb protein by a 60-kDa region of homology called the pocket domain that binds cellular proteins such as E2F and cyclins A and E as well as the viral oncoproteins E1A, E7, and SV40 T antigen. The p107 and Rb proteins have both been implicated as negative regulators of cell growth. We have examined the effect of the unphosphorylated pocket domain of p107 on specific stages of the T antigen-mediated replication of SV40 DNA in vitro. The pocket domain inhibited replication by preventing the assembly of T antigen at the SV40 core origin DNA containing binding site II. However, both proteins formed a ternary complex with DNA containing T antigen-binding site I. The pocket domain of p107 did not inhibit the oligomerization of T antigen in the absence of DNA, and the p107 derivative is bound to the intermediates of this reaction. In the unwinding assay, once T antigen was preassembled as hexamers at the core origin, the pocket domain bound to and stabilized the complex, resulting in an increase in the yield of unwound product. Preformed T antigen hexamers complexed with the pocket domain bind to a synthetic replication fork, and this complex supported unwinding. Consistent with this, the p107 pocket domain had no effect on the helicase activity of T antigen in an assay using a partial duplex substrate. However, a complex containing p107 and T antigen assembled at the core origin did not support SV40 DNA replication in HeLa cell crude extracts or in the monopolymerase reaction. This inhibition is due to the inability of the complex to bind to DNA polymerase alpha, which is required for the initiation of DNA synthesis. The data suggest pathways by which the pocket domain of p107 can negatively regulate T antigen-mediated replication in vitro. The binding of T antigen to p107 is discussed with respect to its role in mitigating negative cell growth control, resulting in viral mediated transformation.
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PMID:Initiation of DNA replication by simian virus 40 T antigen is inhibited by the p107 protein. 812

We have used the T7 DNA replication system to examine coordination of leading and lagging strand synthesis at a replication fork. The 63 kd gene 4 protein provides both helicase and primase activities; we demonstrate that primer synthesis inhibits helicase activity on a synthetic replication fork. Lagging strand DNA synthesis by a complex of gene 4 protein and T7 DNA polymerase decreases the rate of leading strand synthesis. Both leading and lagging strand synthesis are resistant to dilution of the replication proteins, and to challenge with heparin. Furthermore, dilution does not increase the average length of Okazaki fragments. We propose that leading and lagging strand synthesis at a T7 replication fork are coupled and that the replication proteins are recycled.
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PMID:Coordination of leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7. 815 91

During bacteriophage Mu transposition, strand transfer is catalyzed in the presence of phage-encoded A and B proteins and Escherichia coli HU protein, attaching Mu ends to target DNA and creating an intermediate in transposition. Bacteriophage Mu A protein, which remains tightly bound to the Mu ends in the native strand-transfer intermediate, blocked initiation of Mu DNA replication by a system of 8 host proteins (DnaB helicase, DnaC protein, DnaG primase, DNA polymerase III holoenzyme, DNA polymerase I, DNA gyrase, DNA ligase, and single-strand binding protein). This 8-protein system had all enzymatic activities to convert the deproteinized intermediate to a cointegrate; however, additional host factor(s) were required to replicate the native intermediate. While replication of the native intermediate absolutely required DnaB helicase, DnaC protein, and DNA polymerase III holoenzyme, the specific requirements were relaxed for the deproteinized intermediate. Other host factors were able to replace these specific factors. These results indicate that Mu A protein, in conjunction with additional host factor(s), acts to promote assembly of specific host replication proteins at the Mu replication fork. This process may alter the stable interaction of Mu A protein with the ends to allow initiation of Mu DNA synthesis.
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PMID:Participation of the bacteriophage Mu A protein and host factors in the initiation of Mu DNA synthesis in vitro. 820 56

Recently we described the use of human cytomegalovirus (HCMV) cosmid clones in a cotransfection assay of HCMV oriLyt replication (G. S. Pari, M. A. Kacica, and D. G. Anders, J. Virol. 67:2575-2582, 1993). We have now used this assay to identify 11 distinct required loci encoding trans-acting factors sufficient for transient complementation of oriLyt-dependent DNA replication. This set includes all of the virus genes essential to initiate and perform DNA synthesis together with the virus genes required to express these replication functions from their native promoters. Six of the identified loci span open reading frames (ORFs) that encode homologs or probable homologs of herpes simplex virus type 1 replication genes, consistent with predictions based on sequence similarities and biochemical properties. These include the DNA polymerase UL54 and polymerase-associated protein UL44, the single-stranded-DNA-binding protein UL57, and proposed subunits of a helicase-primase complex, UL70, UL105, and UL101-102. Frameshift mutations in any one of these essential ORFs abrogated complementation of DNA replication. Three required loci, UL36-38, IRS1 (or TRS1), and IE1/IE2, encode known regulatory proteins. The remaining two loci span ORFs UL84 and UL112-113 and encode early temporal class nucleus-associated proteins of unknown function. Neither of these genes have been implicated previously in DNA replication or in regulating gene expression, nor have counterparts in herpes simplex virus type 1 or Epstein-Barr virus been described. The results presented here will facilitate investigation of the mechanisms and regulation of HCMV lytic-phase DNA replication.
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PMID:Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA replication. 823 Apr 21


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