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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)

Proliferating cell nuclear antigen (PCNA) is essential for eukaryotic DNA replication and functions as a processivity factor of DNA polymerase delta (pol delta). Due to the functional and structural similarity with the beta-subunit of Escherichia coli DNA polymerase III, it has been proposed that PCNA would act as a molecular clamp during DNA synthesis. By site-directed mutagenesis and biochemical analyses, we have studied the functional domains of human PCNA required for stimulation of replication factor C (RF-C) ATPase and DNA synthesis by pol delta. Short deletions from either the N or C termini caused drastic changes in extraction and chromatographic behaviors, suggesting that both of these terminal regions are crucial to fold the tertiary structure of PCNA. The short C-terminal stretch from Lys254 to Glu256 is necessary for stimulation of RF-C ATPase activity, but not for stimulation of DNA synthesis by pol delta. Nine basic amino acids that are essential for activating DNA synthesis by pol delta are positioned at the internal alpha-helices of PCNA. This result is in good agreement with the observation that PCNA has a ring structure similar to the beta-subunit and clamps a template DNA through this positively charged internal surface. Several other charged amino acids are also required to stimulate either RF-C ATPase or pol delta DNA synthesis. Some of them are positioned at loops which are exposed on one of the side surface of PCNA adjacent to the C-terminal loop. In addition, the beta-sheets composing the intermolecular interface of the trimeric PCNA are important for interaction with pol delta. Therefore, the outer surface of PCNA has multiple functional surfaces which are responsible for the interaction with multiple factors. Furthermore, the two side surfaces seem to be functionally distinguishable, and this may determine the orientation of tracking PCNA along the DNA.
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PMID:Structure-function relationship of the eukaryotic DNA replication factor, proliferating cell nuclear antigen. 767 44

In eukaryotes, nucleotide excision repair of DNA is a complex process that requires many polypeptides to perform dual incision and remove a segment of about 30 nucleotides containing the damage, followed by repair DNA synthesis to replace the excised segment. Nucleotide excision repair DNA synthesis is dependent on proliferating cell nuclear antigen (PCNA). To study gap-filling DNA synthesis during DNA nucleotide excision repair, UV-damaged DNA was first incubated with PCNA-depleted human cell extracts to create repair incisions. Purified DNA polymerase delta or epsilon, with DNA ligase, was then used to form the repair patch. DNA polymerase delta could perform repair synthesis and was strictly dependent on the presence of both PCNA and replication factor C, but gave rise to a very low proportion of complete, ligated circles. The presence of replication protein A (which is also required for nucleotide excision repair) did not alter this result, while addition of DNase IV increased the fraction of ligated products. DNA polymerase epsilon, on the other hand, could fill the repair patch in the absence of PCNA and replication factor C, and most of the products were ligated circles. Addition of replication protein A changed the situation dramatically, and synthesis by polymerase epsilon became dependent on both PCNA and replication factor C. A combination of DNA polymerase epsilon, PCNA, replication factor C, replication protein A, and DNA ligase I appears to be well-suited to the task of creating nucleotide excision repair patches.
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PMID:Nucleotide excision repair DNA synthesis by DNA polymerase epsilon in the presence of PCNA, RFC, and RPA. 771 Oct 23

Complete enzymatic replication of DNA from the simian virus 40 origin has been reconstituted with T antigen and highly purified cellular proteins. DNA polymerase-alpha/primase functions primarily to synthesize RNA-DNA primers for initiation of DNA replication at the origin and for priming each Okazaki fragment. A polymerase switching mechanism requiring replication factor C and the proliferating cell nuclear antigen allows two molecules of DNA polymerase-delta to replicate both strands of the double helix conjointly.
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PMID:Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. 791 Mar 75

To study in details the assembly of DNA polymerases delta and epsilon holoenzymes a circular double-stranded DNA template containing a gap of 45 nucleotides was constructed. Both replication factor C and proliferating cell nuclear antigen were absolutely required and sufficient for assembly of DNA polymerase delta holoenzyme complex on DNA. On such a circular DNA substrate replication protein A (or E. coli single-strand DNA binding protein) was neither required for assembly of DNA polymerase delta holoenzyme complex nor for the gap-filling reaction. A circular structure of the DNA substrate was found to be absolutely critical for the ability of auxiliary proteins to interact with DNA polymerases. The linearization of the circular DNA template resulted in three dramatic effects: (i) DNA synthesis by DNA polymerase delta holoenzyme was abolished, (ii) the inhibition effect of replication factor C and proliferating cell nuclear antigen on DNA polymerase alpha was relieved and (iii) DNA polymerase epsilon could not form any longer a holoenzyme with replication factor C and proliferating cell nuclear antigen. The comparison of the effect of replication factor C and proliferating cell nuclear antigen on DNA polymerases alpha, delta and epsilon indicated that the auxiliary proteins appear to form a mobile clamp, which can easily slide along double-stranded DNA.
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PMID:Assembly of DNA polymerase delta and epsilon holoenzymes depends on the geometry of the DNA template. 791 29

p37 and p40 are two cloned gene products of the five-subunit human cellular DNA replication factor activator 1 (A1) protein complex (also called replication factor C). Here, we describe the solubilization, purification, and characterization of these two proteins that were overproduced in Escherichia coli. Using a nitrocellulose filter binding assay, we demonstrated that the purified A1 p37 protein associated with DNA preferentially at the primer terminus, a property resembling that of the A1 complex. We also show that in the presence of relatively high levels of salt, the recombinant p37 protein alone activated DNA polymerase epsilon but not polymerase delta in catalyzing the elongation of DNA chains. The p40 protein specifically associated with cellular p37 and proliferating-cell nuclear antigen (PCNA) present in HeLa cell cytosolic extract. The addition of purified p40 protein abolished the in vitro polymerase delta-catalyzed DNA elongation reaction dependent on both PCNA and A1. However, this inhibition was reversed by excess polymerase delta, suggesting a specific interaction between the polymerase and the p40 protein. Thus, while p37 binds DNA at the primer end and has a specific affinity for pol epsilon, p40, which binds ATP, interacts with PCNA and pol delta. These activities are essential for the DNA elongation reactions that lead to the synthesis of leading-strand DNA and the maturation of Okazaki fragments.
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PMID:The subunits of activator 1 (replication factor C) carry out multiple functions essential for proliferating-cell nuclear antigen-dependent DNA synthesis. 809 61

The functional interaction of simian virus 40 (SV40) large tumor antigen (T antigen) with DNA polymerase alpha (pol alpha)-primase complex, human single-stranded DNA binding protein (HSSB), and DNA polymerase delta (pol delta) holoenzyme, which includes pol delta, activator I (also called replication factor C), and proliferating cell nuclear antigen, at the replication fork was examined using the purified components that support SV40 DNA replication. Dilution of reaction mixtures during RNA primer synthesis revealed that T antigen remained associated continuously with the fork, while the pol alpha-primase complex dissociated from the complex during oligoribonucleotide synthesis. T antigen unwound duplex DNA from the SV40 core origin at a rate of 200 base pairs/min. Pol alpha-primase complex inhibited the rate of the unwinding reaction, and HSSB, pol alpha, and primase were all required for this effect. These requirements are the same as those essential for DNA primase-catalyzed oligoribonucleotide synthesis (Matsumoto, T., Eki, T., and Hurwitz, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9712-9716). This result suggests that the pol alpha-primase complex interacts with T antigen and HSSB during the unwinding reaction to synthesize RNA primers and that the interaction decreases the rate of T antigen movement. While pol delta holoenzyme can elongate primed DNA chains at a rate of 400-600 nucleotides/min on singly primed phi X174 DNA, the rate of the leading strand synthesis catalyzed by pol delta holoenzyme in the SV40 replication system in vitro was about 200 nucleotides/min. This rate was similar to the unwinding rate catalyzed by T antigen. Thus, the rate of leading strand synthesis catalyzed by pol delta holoenzyme in vitro appears to be limited by the unwinding reaction catalyzed by T antigen.
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PMID:Functional interactions between SV40 T antigen and other replication proteins at the replication fork. 809 7

The identification and purification of human cell proteins required for the production of form I DNA following DNA replication from the simian virus 40 (SV40) origin is described. Using these proteins, complete SV40 DNA replication was reconstituted with only purified DNA replication factors: SV40 large tumor antigen (TAg), replication protein A (RPA), DNA topoisomerases I and II, DNA polymerase alpha-primase, replication factor C (RFC), the proliferating cell nuclear antigen (PCNA), DNA polymerase delta, maturation factor 1 (MF1), and DNA ligase I. MF1, a 5' to 3' exonuclease and DNA ligase I were both identified as essential components for production of covalently closed circular relaxed (form I) DNA. MF1 is probably the same exonuclease previously shown by others to function during DNA synthesis on artificial DNA templates or in conjunction with DNA polymerase alpha from the SV40 origin. Combined with these previous studies, our results suggest that MF1 functions to remove an RNA primer attached to every Okazaki fragment during lagging strand DNA synthesis. Interestingly, whereas mammalian DNA ligase I functioned in the reconstituted replication system, mammalian DNA ligase III did not substitute and the phage T4 DNA ligase functioned inefficiently, suggesting that DNA ligase I has a specific role as a replicative DNA ligase in eukaryotic cells.
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PMID:Reconstitution of complete SV40 DNA replication with purified replication factors. 814 77

The gene encoding the delta' subunit of DNA polymerase III holoenzyme, designated holB, was cloned by a strategy in which peptide sequence was used to derive a DNA hybridization probe. The gene maps to 24.95 centisomes of the chromosome. Sequencing of holB revealed a 1,002-bp open reading frame predicted to produce a 36,936-Da protein. The gene has a ribosome-binding site and promoter that are highly similar to the consensus sequences and is flanked by two potential open reading frames. Protein sequence analysis of delta' revealed a high degree of similarity to the dnaX gene products of Escherichia coli and Bacillus subtilis, including one stretch of 10 identical amino acid residues. A lesser degree of similarity to the gene 44 protein of bacteriophage T4 and the 40-kDa protein of the A1 complex (replication factor C) of HeLa cells was seen. The gene, when placed into a tac promoter-based expression plasmid, directed expression of two proteins of similar size. By immunodetection with anti-holoenzyme immunoglobulin G, both proteins are judged to be products of holB.
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PMID:Identification, isolation, and characterization of the structural gene encoding the delta' subunit of Escherichia coli DNA polymerase III holoenzyme. 850 34

In order to study the effect of trimerization of proliferating cell nuclear antigen (PCNA) on its interaction with DNA polymerase (pol) delta and its loading onto DNA by replication factor C (RF-C) we have mutated a single tyrosine residue located at the subunit interface (Tyr114) to alanine. This mutation (Y114A) had a profound effect on PCNA, since it completely abolished trimer formation as seen by glycerol gradient sedimentation and native gel electrophoresis. Furthermore, the mutant protein was unable to stimulate DNA synthesis by pol delta and did not compete effectively with wild-type PCNA for pol delta, although it was able to oligomerize and could to some extent interact with subunits of functionally active PCNA. We thus conclude that PCNA molecules that are not part of a circular trimeric complex cannot interact with the pol delta core. furthermore, the mutant protein could not be loaded onto DNA by RF-C and did not compete with wild-type PCNA for loading onto DNA, indicating that PCNA trimerization may also be a prerequisite for its recognition by RF-C. The adverse effects caused by this single mutation suggest that trimerization of PCNA is essential for the monomers to keep their overall structure and that the structural changes imposed by trimerization are important for interaction with other proteins.
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PMID:Tyrosine 114 is essential for the trimeric structure and the functional activities of human proliferating cell nuclear antigen. 852 31

By using a complementation assay for a replication factor C dependent DNA polymerase activity on a singly-primed M13 DNA template, we have isolated from calf thymus a multiprotein complex active in DNA replication. For this, the inclusion of ATP during the entire isolation procedure was essential, since the complex decayed after omission of ATP. This complex contains at least DNA polymerase alpha/primase, DNA polymerase delta, and replication factor C as shown by gel-filtration and coimmunoprecipitation experiments. It is functionally active in replication of primed and unprimed single-stranded M13 DNA templates. Furthermore, in the presence of proliferating cell nuclear antigen and ATP, it forms an isolatable holoenzyme/template-primer complex. Replication factor C apparently mediates the interaction of DNA polymerase delta in the complex with proliferating cell nuclear antigen, through an ATP-dependent mechanism. This interaction appears to stabilize the binding of the complex to a template-primer and to coordinate the activity of DNA polymerase alpha/primase and DNA polymerase delta during replication of a single-stranded DNA template. Our data suggest the existence of an asymmetric DNA polymerase complex in mammalian cells.
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PMID:DNA replication machinery: functional characterization of a complex containing DNA polymerase alpha, DNA polymerase delta, and replication factor C suggests an asymmetric DNA polymerase dimer. 863 37


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