Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Interactions between the herpes simplex virus type 1 (HSV-1) origin (ori)-binding protein (UL9) and two other components of the functional DNA replication complex have been observed. However, to date, no interaction between UL9 and a component of the DNA polymerase holoenzyme has been demonstrated. In this report, we demonstrate that UL9 and the DNA polymerase accessory protein (UL42) can form a stable complex in vitro as determined by coimmunoprecipitation with specific antibodies to each protein and by affinity chromatography using glutathione S-transferase (GST) fusion proteins. Complex formation does not require the presence of other viral proteins and occurs in the presence of ethidium bromide, indicating that UL9-UL42 interaction is DNA independent. Affinity beads charged with increasing concentrations of GST-42 fusion protein up to 5 microM bound increasing amounts of UL9 expressed by in vitro transcription/translation in rabbit reticulocyte lysates. Binding of N- and C-terminal portions of UL9 to GST affinity matrices revealed that the N-terminal 533 amino acids were sufficient for binding to GST-42, albeit at approximately a four- to six-fold reduced affinity compared to the full-length protein. No binding of a polypeptide containing the remainder of the UL9 C-terminal residues was observed. Thus the ori-binding protein, UL9, can physically associate with at least one member of each of the complexes (helicase/primase, DNA polymerase holoenzyme, single-stranded DNA-binding protein) required for origin-dependent DNA replication. These specific interactions provide a means by which the ordered assembly of HSV-1 DNA replication proteins at origins of replication can occur in the infected cell for initiation of viral DNA synthesis.
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PMID:Interaction between the herpes simplex virus type 1 origin-binding and DNA polymerase accessory proteins. 945 23

The gene 2.5 single-stranded DNA (ssDNA) binding protein of bacteriophage T7 is essential for T7 DNA replication and recombination. Earlier studies have shown that the COOH-terminal 21 amino acids of the gene 2.5 protein are essential for specific protein-protein interaction with T7 DNA polymerase and T7 DNA helicase/primase. A truncated gene 2.5 protein, in which the acidic COOH-terminal 21 amino acid residues are deleted no longer supports T7 growth, forms dimers, or interacts with either T7 DNA polymerase or T7 helicase/primase in vitro. The single-stranded DNA-binding protein encoded by Escherichia coli (SSB protein) and phage T4 (gene 32 protein) also have acidic COOH-terminal domains, but neither protein can substitute for T7 gene 2.5 protein in vivo. To determine if the specificity for the protein-protein interaction involving gene 2.5 protein resides in its COOH terminus, we replaced the COOH-terminal region of the gene 2.5 protein with the COOH-terminal region from either E. coli SSB protein or T4 gene 32 protein. Both of the two chimeric proteins can substitute for T7 gene 2.5 protein to support the growth of phage T7. The two chimeric proteins, like gene 2.5 protein, form dimers and interact with T7 DNA polymerase and helicase/primase to stimulate their activities. In contrast, chimeric proteins in which the COOH terminus of T7 gene 2.5 protein replaced the COOH terminus of E. coli SSB protein or T4 gene 32 protein cannot support the growth of phage T7. We conclude that an acidic COOH terminus of the gene 2.5 protein is essential for protein-protein interaction, but it alone cannot account for the specificity of the interaction.
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PMID:Role of the acidic carboxyl-terminal domain of the single-stranded DNA-binding protein of bacteriophage T7 in specific protein-protein interactions. 949 92

Escherichia coli DNA polymerase III holoenzyme contains 10 different subunits which assort into three functional components: a core catalytic unit containing DNA polymerase activity, the beta sliding clamp that encircles DNA for processive replication, and a multisubunit clamp loader apparatus called gamma complex that uses ATP to assemble the beta clamp onto DNA. We examine here the function of the psi subunit of the gamma complex clamp loader. Omission of psi from the holoenzyme prevents contact with single-stranded DNA-binding protein (SSB) and lowers the efficiency of clamp loading and chain elongation under conditions of elevated salt. We also show that the product of a classic point mutant of SSB, SSB-113, lacks strong affinity for psi and is defective in promoting clamp loading and processive replication at elevated ionic strength. SSB-113 carries a single amino acid replacement at the penultimate residue of the C-terminus, indicating the C-terminus as a site of interaction with psi. Indeed, a peptide of the 15 C-terminal residues of SSB is sufficient to bind to psi. These results establish a role for the psi subunit in contacting SSB, thus enhancing the clamp loading and processivity of synthesis of the holoenzyme, presumably by helping to localize the holoenzyme to sites of SSB-coated ssDNA.
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PMID:Devoted to the lagging strand-the subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly. 954 54

The coordinated synthesis of both leading and lagging DNA strands is thought to involve a dimeric DNA polymerase and a looping of the lagging strand so that both strands can be synthesized in the same direction. We have constructed a minicircle with a replication fork that permits an assessment of the stoichiometry of the proteins and a measurement of the synthesis of each strand. The replisome consisting of bacteriophage T7 DNA polymerase, helicase, primase, and single-stranded DNA-binding protein mediates coordinated replication. The criteria for coordination are fulfilled: (1) a replication loop is formed, (2) leading and lagging strand synthesis are coupled, (3) the lagging strand polymerase recycles from one Okazaki fragment to another, and (4) the length of Okazaki fragments is regulated. T7 single-stranded DNA-binding protein is essential for coordination.
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PMID:Coordinated leading and lagging strand DNA synthesis on a minicircular template. 965 83

A complex of the chi and psi proteins is required to confer resistance to high levels of glutamate on the DNA polymerase III holoenzyme-catalyzed reaction (Olson, M., Dallmann, H. G., and McHenry, C. (1995) J. Biol. Chem. 270, 29570-29577). We demonstrate that this salt resistance also requires templates to be coated with the Escherichia coli single-stranded DNA-binding protein (SSB). We show that this is the result of a direct chipsi-SSB interaction that is strengthened approximately 1000-fold when SSB is bound to DNA. On model oligonucleotide templates, DNA polymerase III core is inhibited by SSB. We show that the minimal polymerase assembly that will synthesize DNA on SSB-coated templates is polymerase III-tau-psi chi. gamma, the alternative product of the dnaX gene, will not replace tau in this reaction, indicating that tau's unique ability to bind to DNA polymerase III holding chipsi in the same complex is essential. All of our findings are consistent with chipsi strengthening DNA polymerase III holoenzyme interactions with the SSB-coated lagging strand at the replication fork, facilitating complex assembly and elongation.
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PMID:The chi psi subunits of DNA polymerase III holoenzyme bind to single-stranded DNA-binding protein (SSB) and facilitate replication of an SSB-coated template. 972 85

Replication of the two template strands at eukaryotic cell DNA replication forks is a highly coordinated process that ensures accurate and efficient genome duplication. Biochemical studies, principally of plasmid DNAs containing the Simian Virus 40 origin of DNA replication, and yeast genetic studies have uncovered the fundamental mechanisms of replication fork progression. At least two different DNA polymerases, a single-stranded DNA-binding protein, a clamp-loading complex, and a polymerase clamp combine to replicate DNA. Okazaki fragment synthesis involves a DNA polymerase-switching mechanism, and maturation occurs by the recruitment of specific nucleases, a helicase, and a ligase. The process of DNA replication is also coupled to cell-cycle progression and to DNA repair to maintain genome integrity.
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PMID:The DNA replication fork in eukaryotic cells. 975 2

The DNA polymerase (DNApol) of Autographa californica nuclear polyhedrosis virus was purified to homogeneity from recombinant baculovirus-infected cells. DNApol was active in polymerase assays on singly primed M13 template, and full-length replicative form II product was synthesized at equimolar ratios of enzyme to template. The purified recombinant DNApol was shown to be processive by template challenge assay. Furthermore, DNApol was able to incorporate hundreds of nucleotides on an oligo(dT)-primed poly(dA) template with limiting amounts of polymerase. DNApol has moderate strand displacement activity, as it was active on nicked and gapped templates, and displaced a primer in a replication-dependent manner. Addition of saturating amounts of LEF-3, the viral single-stranded DNA-binding protein (SSB), increased the innate strand displacement ability of DNApol. However, when LEF-3 was added prior to the polymerase, it failed to stimulate DNApol replication on a singly primed M13 template because the helix-destabilizing activity of LEF-3 caused the primer to dissociate from the template. Escherichia coli SSB efficiently substituted for LEF-3 in the replication of a nicked template, suggesting that specific protein-protein interactions were not required for strand displacement in this assay.
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PMID:Autographa californica nuclear polyhedrosis virus DNA polymerase: measurements of processivity and strand displacement. 1023 52

Functional interactions between mitochondrial DNA polymerase (pol gamma) and mitochondrial single-stranded DNA-binding protein (mtSSB) from Drosophila embryos have been evaluated with regard to the overall activity of pol gamma and in partial reactions involving template-primer binding and initiation and idling in DNA strand synthesis. Both the 5' --> 3' DNA polymerase and 3' --> 5' exonuclease in pol gamma are stimulated 15-20-fold on oligonucleotide-primed single-stranded DNA by native and recombinant forms of mtSSB. That the extent of stimulation is similar for both enzyme activities over a broad range of KCl concentrations suggests their functional coordination and a similar mechanism of stimulation by mtSSB. At the same time, the high mispair specificity of pol gamma in exonucleolytic hydrolysis is maintained, indicating that enhancement of pol gamma catalytic efficiency is likely not accompanied by increased nucleotide turnover. DNase I footprinting of pol gamma.DNA complexes and initial rate measurements show that mtSSB enhances primer recognition and binding and stimulates 30-fold the rate of initiation of DNA strands. Dissociation studies show that productive complexes of the native pol gamma heterodimer with template-primer DNA are formed and remain stable in the absence of replication accessory proteins.
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PMID:Functional interactions of mitochondrial DNA polymerase and single-stranded DNA-binding protein. Template-primer DNA binding and initiation and elongation of DNA strand synthesis. 1032 75

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, Phi29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from Phi29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3' --> 5' exonuclease domain (DNA polymerase II exo(-) and T7 pol exo(-)) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.
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PMID:Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency. 1048 82

Drosophila mitochondrial DNA polymerase has been reconstituted and purified from baculovirus-infected insect cells. Baculoviruses encoding full-length and mature forms of the catalytic and accessory subunits were generated and used in single and co-infection studies. Recombinant heterodimeric holoenzyme was reconstituted in both the mitochondria and cytoplasm of Sf9 cells and required the mitochondrial presequences in both subunits. The recombinant holoenzyme contains DNA polymerase and 3'-5' exonuclease that are stimulated substantially by both salt and mitochondrial single-stranded DNA-binding protein. Thus, the recombinant enzyme exhibits biochemical properties indistinguishable from those of the native enzyme from Drosophila embryos. Production of the catalytic subunit alone yielded soluble protein with the chromatographic properties of the heterodimeric holoenzyme. However, the purified catalytic core has a 50-fold lower specific activity. This provides evidence of a critical role for the accessory subunit in the catalytic efficiency of Drosophila mitochondrial DNA polymerase.
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PMID:Baculovirus expression reconstitutes Drosophila mitochondrial DNA polymerase. 1050 44


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