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)

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.
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PMID:The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins. 194 34

DNA polymerase alpha and primase are two key enzymatic components of the eukaryotic DNA replication complex. In situ hybridization of cloned cDNAs for mouse DNA polymerase alpha and for the two subunits of mouse primase has been utilized to physically map these genes in the mouse genome. The DNA polymerase alpha gene (Pola) was mapped to the mouse X chromosome in region C-D. The gene encoding the p58 subunit of primase (Prim2) was located to mouse chromosome 1 in region A5-B and the p49 subunit gene (Prim1) was found to be on mouse chromosome 10 in the distal part of band D that is close to the telomere. Current knowledge of mouse and human conserved chromosomal regions along with the findings presented here lead to predictions of where the genes for the DNA primase subunits may be found in the human genome: the p58 subunit gene may be on human chromosome 2 and the p49 subunit gene on human chromosome 12. The mapping of Pola to region C-D of the mouse X chromosome adds a new marker in a conserved region between the mouse X chromosome and region Xp21-22.1 of the human X chromosome.
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PMID:Physical mapping of the genes for three components of the mouse DNA replication complex: polymerase alpha to the X chromosome, primase p49 subunit to chromosome 10, and primase p58 subunit to chromosome 1. 203 91

The immunoaffinity-purified subunits of the yeast DNA primase-DNA polymerase protein complex and subunit-specific monoclonal antibodies were used to explore the structural relationships of the subunits in the complex. The reconstituted four-subunit complex (180-, 86-, 58-, and 49-kDa polypeptides) behaved as a single species, exhibiting a Stokes radius of 80 A and a sedimentation coefficient of 8.9 S. The calculated molecular weight of the reconstituted complex is 312,000. We infer that the stoichiometry of the complex is one of each subunit per complex. The complex has a prolate ellipsoid shape with an axial ratio of approximately 16. When the 180-kDa and DNA primase subunits were recombined in the absence of the 86-kDa subunit, a physical complex formed, as judged by immunoprecipitation of DNA primase activity and polypeptides with an anti-180-kDa monoclonal antibody. While the 86-kDa subunit readily forms a physical complex with the 180-kDa DNA polymerase catalytic subunit, we have not detected a complex containing 86-kDa and the DNA primase subcomplex (49- and 58-kDa subunits). The 86-kDa subunit was not required for DNA primase-DNA polymerase complex formation; the 180-kDa subunit and DNA primase heterodimer directly interact. However, the presence of the 86-kDa subunit increased the rate at which the DNA primase and 180-kDa polypeptides formed a complex and increased the total fraction of DNA primase activity that was associated with DNA polymerase activity. The observations demonstrate that the DNA primase p49.p58 heterodimer and the DNA polymerase p86.p180 heterodimer interact via the 180-kDa subunit. The four-subunit reconstituted complex was sufficient to catalyze the DNA chain extension coupled to RNA primer synthesis on a single-stranded DNA template, as previously observed in the conventionally purified complex isolated from wild type cells.
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PMID:Reconstitution of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex in vitro. The 86-kDa subunit facilitates but is not required for complex formation. 203 67

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.
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PMID:Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells. 242 27

Primase is a specialized RNA polymerase that synthesizes RNA primers for initiation of DNA synthesis. A full cDNA clone of the p49 subunit of mouse primase, a heterodimeric enzyme, has been isolated using a primase p49-specific polyclonal antibody to screen a lambda gt11 mouse cDNA expression library. The cDNA indicated the subunit is a 417-amino acid polypeptide with a calculated molecular mass of 49,295 daltons. The p49 mRNA is approximately 1500 nucleotides long with a 5'-untranslated region of 74 nucleotides and a 3'-untranslated region of 200 nucleotides. Comparison with a similar sized primase subunit from yeast showed highly conserved amino acid sequences in the N-terminal halves of the polypeptides and included a potential metal-binding domain suggesting the functional importance of this region for DNA binding. In contrast, the 3' portion of the cDNA has rapidly diverged in nucleotide sequence, as primase mRNA can be detected in mouse and rat cells with a 3' probe (including coding and noncoding) but not in RNA from hamster or human cells. A full-length cDNA probe detected mRNA from hamster and human cell lines, indicating a conserved 5' portion and divergent 3' region of the expressed gene. The rapid divergence may be related to the species-specific protein interactions found for the DNA polymerase alpha-primase complex. The mRNA is detected in proliferating but not in quiescent cells consistent with its function in DNA replication.
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PMID:Mouse primase p49 subunit molecular cloning indicates conserved and divergent regions. 292 77

In the eukaryotic cell, DNA synthesis is initiated by DNA primase associated with DNA polymerase alpha. The eukaryotic primase is composed of two subunits, p49 and p58, where the p49 subunit contains the catalytic active site. Mutagenesis of the cDNA for the p49 subunit was initiated to demonstrate a functional correlation of conserved residues among the eukaryotic primases and DNA polymerases. Fourteen invariant charged residues in the smaller catalytic mouse primase subunit, p49, were changed to alanine. These mutant proteins were expressed, purified, and enzymatically characterized for primer synthesis. Analyses of the mutant proteins indicate that residues 104-111 are most critical for primer synthesis and form part of the active site. Alanine substitution in residues Glu105, Asp109, and Asp111 produced protein with no detectable activity in direct primase assays, indicating that these residues may form part of a conserved carboxylic triad also observed in the active sites of DNA polymerases and reverse transcriptases. All other mutant proteins showed a dramatic decrease in catalysis, while mutation of two residues, Arg162 and Arg163, caused an increase in Km(NTP). Analysis of these mutant proteins in specific assays designed to separately investigate dinucleotide formation (initiation) and elongation of primer indicates that these two activities utilize the same active site within the p49 subunit. Finally, mutations in three active site codons produced protein with reduced affinity with the p58 subunit, suggesting that p58 may interact directly with active site residues.
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PMID:Active site mapping of the catalytic mouse primase subunit by alanine scanning mutagenesis. 787 36

We have studied the expression of genes encoding DNA replication proteins during different cell growth events. Gene expression of human DNA polymerase alpha-DNA primase, a principal chromosomal replication enzyme complex, is up-regulated during the entrance of a cell from quiescence into the mitotic cell cycle. In contrast, expression of these genes is greatly reduced in fibroblasts rendered temporarily quiescent by contact inhibition or serum starvation. In actively cycling cells, DNA polymerase alpha-DNA primase genes are expressed at all stages of the cell cycle. To investigate how their gene expression is regulated in cells permanently exiting the cell cycle during terminal differentiation, we used a novel method to obtain a pure population of such cells. In this report, we describe the down-regulation of gene expression of DNA polymerase alpha during both HL-60 (human myeloid) and MEL (mouse erythroleukemia) cell differentiation. Gene expression of the two subunits of DNA primase, p49 and p58, is also down-regulated at the mRNA level in differentiated MEL cells. In differentiated HL-60 cells, the decline of DNA polymerase alpha gene expression occurs at both the transcript and protein levels. Down-regulation of DNA polymerase alpha at the steady state transcript level is caused, at least in part, by a decreased rate of transcription initiation without transcription elongation block.
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PMID:Down-regulation of genes encoding DNA replication proteins during cell cycle exit. 804 55

The enzymatic mechanism of primase was investigated using Escherichia coli and baculoviral overexpressed mouse primase subunits, p49 and p58. Neither of the singly purified primase subunits displayed primase activity alone, but the p49 subunit was able to extend a riboprimer, indicating that this subunit contains an RNA polymerase activity. The p58 subunit cooperated with the p49 subunit in binding the initiating purine to form the initial dinucleotide. After initiation, the p49 subunit alone was sufficient to extend the growing primer, but both the rate of p49 primer extension and its stability were influenced by the p58 subunit. The Km(ATP) in primer synthesis on poly(dT) of the p49-p58 heterodimeric primase complex was 10-fold higher than the Km(ATP) of the single p49 subunit in a ribo(A) primer extension assay. In addition, labeled ATP cross-linked to both of the individually purified subunits but with a striking difference in affinities; cross-linking was 11-fold more efficient to the p49 subunit. The interaction of the two primase subunits with polymerase alpha was also investigated. Immunoprecipitation experiments indicate that only the p58 subunit directly contacts the p180 subunit of DNA polymerase alpha. Competition experiments in the coupled primase-polymerase assay with a catalytically inactive mutant of DNA polymerase alpha and the Klenow fragment suggest that the DNA polymerase alpha-primase complex does not dissociate from the primer during the transition from RNA to DNA synthesis.
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PMID:Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication. 825 37

Primase plays a key role in governing the sequence of events required on the lagging strand during a cycle of Okazaki fragment synthesis. To begin to probe the protein-protein interactions necessary for primase function at the replication fork, we have used limited trypsinolysis to separate primase into two functional domains, an N-terminal domain of 49 kDa (p49) and a carboxyl-terminal domain of 16 kDa (p16). p49 retained primase activity in replication assays that utilized bacteriophage M13 DNA carrying the bacteriophage G4 origin of DNA replication as the template, but was inactive during general priming or the conversion of phi X174 single-stranded circular (ss(c))-DNA to the replicative form (RF) and could not support lagging-strand DNA synthesis at replication forks reconstituted with the phi X-type primosomal proteins and the DNA polymerase III holoenzyme. On the other hand, p16 inhibited those replication reactions that included the replication fork helicase, DnaB (general priming, phi X174 ss(c)-->RF, and at the replication fork), but had no effect on those that did not (M13Gori ss(c)-->RF). These results demonstrate that p49 defines a domain of primase required for catalytic activity, that p16 defines a domain of primase required for functional interaction with DnaB, and that it is a protein-protein interaction with DnaB that attracts primase to the replication fork.
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PMID:Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork. 830 39

Comparison of the amino acid sequences of eucaryotic DNA primase and the family X polymerases indicates that primase shares significant sequence homology with this family. With the use of DNA polymerase beta (pol beta) as a paradigm for family X polymerases, these homologies include both the catalytic core domain/subunit of each enzyme (31 kDa domain of pol beta and p49 subunit of primase) as well as the accessory domain/subunit (8 kDa domain of pol beta and p58 subunit of primase). To further explore these homologies as well as provide insights into the mechanism of primase, we generated three mutants (R304K, R304Q, and R304A) of the p49 subunit at an arginine that is highly conserved between primase and the eukaryotic family X polymerases. These mutations significantly decreased the rate of primer synthesis, due primarily to a decreased rate of initiation, and the extent of impairment correlated with the severity of the mutation (A > Q > K). R304 also contributes to efficient utilization of the NTP that will become the 5'-terminus of the new primer, and these effects are at least partially mediated through interactions with the phosphates of this NTP. The implications of these results with respect to the structure and biological role of primase, as well as its relationship to the family X polymerases, are discussed.
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PMID:Arg304 of human DNA primase is a key contributor to catalysis and NTP binding: primase and the family X polymerases share significant sequence homology. 1038 12


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