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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 amino terminus of the E2F1 transcription factor is a protein-protein interaction domain since it associates with cyclin A/cdk2. Here, the two-hybrid yeast screen was used to clone genes whose products associate with the amino terminus of E2F1. The amino-terminal 121 amino acids of E2F1 were fused to the Lex A binding domain while a partial length cDNA library from the embryo of a 12 day old mouse was fused to the VP16 activation domain. Following coexpression of these fusions in yeast, two novel genes were cloned that code for proteins that associate with E2F1. In an in vitro assay, these E2F1 Binding Proteins (EBP1 and EBP2) associate with residues 1-121 of E2F1 or with the full-length protein; however, they do not associate with its carboxy terminus (residues 88-437). When EBP1 or EBP2 were expressed in COS cells along with E2F1 and the target promoter DNA polymerase alpha, repression of transcription was observed. However, no repression of DNA polymerase alpha was seen if the cells expressed a nonassociating mutant E2F1 (residues 88-437), along with EBP1 or EBP2. Finally, expression of the EBP2 gene is up-regulated in growing NIH3T3 fibroblasts, relative to serum-starved cells. However, this up-regulation of EBP2 expression is not seen in fibroblasts constitutively expressing E2F1.
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PMID:Isolation of two novel cDNAs whose products associate with the amino terminus of the E2F1 transcription factor. 882 66

In Saccharomyces cerevisiae, the CDC2 gene encodes the large subunit of DNA polymerase III, the analogue of mammalian DNA polymerase delta. We have isolated DNA fragments from a library of Candida albicans genomic DNA in the vector pRS316 that rescue temperature sensitive cdc2 mutations in S. cerevisiae. These fragments contain an ORF coding for a protein of 1038 aa with a predicted molecular mass of 118.8 kDa. The predicted protein shows homology to a number of eukaryotic DNA polymerases, with 62% identity over its length to the S. cerevisiae Cdc2 protein. It also contains a number of motifs which are characteristic of DNA polymerases in general and viral polymerases in particular, as well as the conserved motif which interacts with proliferating cell nuclear antigen. These results indicate that this gene is C. albicans POL3. Analysis of the expression of C. albicans POL3 revealed that the transcript is present throughout the mitotic cell cycle, which contrasts with the expression of S. cerevisiae CDC2.
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PMID:Isolation and molecular characterisation of the POL3 gene from Candida albicans. 899 2

In recent years, work from a large number of laboratories has greatly expanded our knowledge of the biochemical characteristics and the genetic structure of the DNA polymerases used during papovavirus DNA replication. The development of in vitro DNA replication systems for both SV40 and polyoma virus has been paramount in facilitating the development of the current models describing how DNA polymerase alpha and delta function to replicate the genomes of these two viruses. Our studies have demonstrated that the proteins recognized to be essential for both in vitro SV40 and polyoma viral origin-dependent DNA synthesis can be isolated from cells as an intact complex. We have shown that the human cell MRC closely resembles the murine cell MRC, in both its protein composition and its fractionation and chromatographic profile. In addition, our data regarding both the human and the murine MRC support the dipolymerase model proposed from in vitro DNA replication studies using reconstituted assay systems. In addition, analysis of the nucleotide sequence of the genes encoding DNA polymerase alpha and delta has revealed that the amino acids encoded by several regions of these two genes have been rigorously maintained across evolutionary lines. This information has permitted the identification of protein domains which mediate the complex series of protein-protein interactions that direct the DNA polymerases to the cell nucleus, specify complete or partial exonuclease active sites, and participate in the interaction of each DNA polymerase with the DNA template. Expression studies examining each of the genes encoding DNA polymerase alpha and delta clearly indicate that both DNA polymerases are cell cycle regulated and undergo a dramatic induction in their expression when quiescent cells are stimulated to enter the cell cycle. This is in contrast to the two- to three-fold upregulation in the level of expression of these two genes when cycling cells cross the G1/S boundary. In addition, both proteins are phosphorylated in a cell cycle-dependent manner, and phosphorylation appears to be mediated through the action of a cdc2-dependent protein kinase. Despite all of this new information, much remains to be learned about how papovavirus DNA replication is regulated and how these two DNA polymerases act in vivo to faithfully copy the viral genomes. Studies have yet to be performed which identify all of the cellular factors which potentially mediate papovavirus DNA replication. The reconstituted replication systems have yielded a minimum number of proteins which are required to replicate SV40 and polyoma viral genomes in vitro. However, further studies are needed to identify additional factors which may participate in each step of the initiation, elongation, and termination phases of viral genome replication. As an example, models describing the potential role of cellular helicases, which are components of the MRC isolated from murine and human cells, have yet to be described. It is also conceivable that there are a number of other proteins which serve to attach the MRC to the nuclear matrix, stimulate viral DNA replication, and potentially regulate various aspects of the activity of the MRC throughout viral DNA replication. We are currently working toward characterizing the biochemical composition of the MRC from both murine and human cells. Our goals are to identify all of the structural components of the MRC and to define the role of these components in regulating papovavirus and cellular DNA replication. We have also begun studies to visualize the spatial organization of these protein components within the MRC, examine the regulatory processes controlling the activity of the various components of the MRC, and then develop this information into a coherent picture of the higher order structure of the MRC within the cell nucleus. We believe that this information will enable us to develop an accurate view of the detailed processes mediating both pa
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PMID:Expression, purification, and characterization of DNA polymerases involved in papovavirus replication. 902 36

In temperature-sensitive (ts) mutants of mouse FM3A cells, the levels of mutagenesis and survival of cells treated with DNA-damaging agents have been difficult to assess because they are killed after their mutant phenotypes are expressed at the nonpermissive temperature. To avoid this difficulty, we incubated the ts mutant cells at the restrictive temperature, 39 degrees C, for only a limited period after inducing DNA damage. We used ts mutants defective in genes for ubiquitin-activating enzyme (E1), DNA polymerase alpha, and p34(cdc2) kinase. Whereas the latter two showed no effect, E1 mutants were sensitized remarkably to UV light if incubated at 39 degrees C for limited periods after UV exposure. Eighty-five percent of the sensitization occurred within the first 12 h of incubation at 39 degrees C, and more than 36 h at 39 degrees C did not produce any further sensitization. Moreover, while the 39 degrees C incubation gave E1 mutants a moderate spontaneous mutator phenotype, the same treatment significantly diminished the level of UV-induced 6-thioguanine resistance mutagenesis and extended the time necessary for expression of the mutation phenotype. These characteristics of E1 mutants are reminiscent of the defective DNA repair phenotypes of Saccharomyces cerevisiae rad6 mutants, which have defects in a ubiquitin-conjugating enzyme (E2), to which E1 is known to transfer ubiquitin. These results demonstrate the involvement of E1 in eukaryotic DNA repair and mutagenesis and provide the first direct evidence that the ubiquitin-conjugation system contributes to DNA repair in mammalian cells.
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PMID:Incubation at the nonpermissive temperature induces deficiencies in UV resistance and mutagenesis in mouse mutant cells expressing a temperature-sensitive ubiquitin-activating enzyme (E1). 903 76

DNA polymerase alpha-primase is the only known eukaryotic enzyme that can start DNA replication de novo. In this study, we investigated the regulation of DNA replication by phosphorylation of DNA polymerase alpha-primase. The p180 and the p68 subunits of DNA polymerase alpha-primase were phosphorylated using Cyclin A-, B- and E- dependent kinases. This phosphorylation did not influence its DNA polymerase activity on activated DNA, but slightly stimulated primase activity using poly(dT) single-stranded DNA (ssDNA) without changing the product length of primers. In contrast, site-specific initiation of replication on plasmid DNA containing the SV40 origin is affected: Cyclin A-Cdk2 and Cyclin A-Cdc2 inhibited initiation of SV40 DNA replication in vitro, Cyclin B-Cdc2 had no effect and Cyclin E-Cdk2 stimulated the initiation reaction. DNA polymerase alpha-primase that was pre-phosphorylated by Cyclin A-Cdk2 was completely unable to initiate the SV40 DNA replication in vitro; Cyclin B-Cdc2-phosphorylated enzyme was moderately inhibited, while Cyclin E-Cdk2-treated DNA polymerase alpha-primase remained fully active in the initiation reaction.
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PMID:Phosphorylation of DNA polymerase alpha-primase by cyclin A-dependent kinases regulates initiation of DNA replication in vitro. 912 53

The catalytic subunit of human DNA polymerase (pol) delta was overexpressed in an active, soluble form by the use of a baculovirus system in insect cells. The recombinant enzyme was separated from endogenous DNA polymerases by phosphocellulose, Mono Q-Sepharose, and single-stranded DNA-cellulose chromatography. Recombinant DNA pol delta was also purified by immunoaffinity chromatography. The enzymatic properties of the purified catalytic subunit were characterized. The enzyme was active and possessed both DNA polymerase and associated 3' to 5' exonuclease activities. NH2-terminal deletion mutants retained polymerase activity, whereas the core and COOH-terminal deletion mutants were devoid of any measurable activities. Coinfection of Sf9 cells with recombinant baculovirus vectors for pol delta and cyclin-dependent kinase (cdk)-cyclins followed by metabolic labeling with 32Pi showed that the recombinant catalytic subunit of pol delta could be hyperphosphorylated by G1 phase-specific cdk-cyclins. When cdk2 was coexpressed with pol delta in Sf9 cells, pol delta was found to coimmunoprecipitate with antibodies against cdk2. Experiments with deletion mutants of pol delta showed that the NH2-terminal region was essential for this interaction. Coimmunoprecipitation and Western blot experiments in Molt 4 cells confirmed the interaction in vivo. Preliminary experiments showed that phosphorylation of the catalytic subunit of pol delta by cdk2-cyclins had little or no effect on the specific activity of the enzyme.
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PMID:Characterization of the p125 subunit of human DNA polymerase delta and its deletion mutants. Interaction with cyclin-dependent kinase-cyclins. 954 86

Eukaryotic DNA replication is limited to once per cell cycle because cyclin-dependent kinases (cdks), which are required to fire origins, also prevent re-replication. Components of the replication apparatus, therefore, are 'reset' by cdk inactivation at the end of mitosis. In budding yeast, assembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) at origins can only occur during G1 because it is blocked by cdk1 (Cdc28) together with B cyclins (Clbs). Here we describe a second, separate process which is also blocked by Cdc28/Clb kinase and, therefore, can only occur during G1; the recruitment of DNA polymerase alpha-primase (pol alpha) to chromatin. The recruitment of pol alpha to chromatin during G1 is independent of pre-RC formation since it can occur in the absence of Cdc6 protein. Paradoxically, overproduction of Cdc6p can drive both dephosphorylation and chromatin association of pol alpha. Overproduction of a mutant in which the N-terminus of Cdc6 has been deleted is unable to drive pol alpha chromatin binding. Since this mutant is still competent for pre-RC formation and DNA replication, we suggest that Cdc6p overproduction resets pol alpha chromatin binding by a mechanism which is independent of that used in pre-RC assembly.
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PMID:Evidence for a Cdc6p-independent mitotic resetting event involving DNA polymerase alpha. 967 28

DNA polymerase alpha-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase alpha-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase alpha-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase alpha-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase alpha-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.
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PMID:Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation. 985 88

Telomerase activity was measured at each phase of the cell cycle in synchronized tobacco (Nicotiana tabacum) BY-2 cells in suspension culture with the use of the telomeric repeat amplification protocol assay. The activity was low or undetectable at most phases of the cell cycle but showed a marked increase at early S phase. The induction of telomerase activity was not affected by the S phase blockers aphidicolin (which inhibits DNA polymerase alpha) or hydroxyurea (which inhibits ribonucleotide reductase), but it was prevented by olomoucine, an inhibitor of Cdc2/Cdk2 kinases that blocks G(1)-S cell cycle transition. These results suggest that the induction of telomerase activity is not directly coupled to DNA replication by conventional DNA polymerases, but rather is triggered by the entry of cells into S phase. Various analogs of the plant hormone auxin, including indole-3-acetic acid, alpha-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid, potentiated the increase in telomerase activity at early S phase; the growth-inactive analog 2,3-dichlorophenoxyacetic acid, however, had no such effect. Potentiation by indole-3-acetic acid of the induction of telomerase activity was dose dependent. Together, these data indicate that telomerase activity in tobacco cells is regulated in a cell cycle-dependent manner, and that the increase in activity at S phase is specifically inducible by auxin.
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PMID:Auxin induction of cell cycle regulated activity of tobacco telomerase. 1040 48

PCNA (proliferating cell nuclear antigen), originally characterized as a DNA polymerase accessory protein, functions as a DNA sliding clamp for DNA polymerase delta and is an essential component for eukaryotic chromosomal DNA replication. Recent studies have revealed a striking feature of PCNA in its ability to interact with multiple partners, involved, for example, in Okazaki fragment joining, DNA repair, DNA methylation and chromatin assembly. Since these reactions take place mainly on replicating DNA, PCNA has applications as a marker for DNA synthesis. It is of interest that proteins involved in cell cycle regulation may also exhibit PCNA binding activity. For example, the CDK inhibitor, p21 (Cip1/Waf1) interacts with PCNA blocking its activity necessary for DNA replication and also affecting interactions with other PCNA binding proteins. The available data indicate that DNA sliding clamps have generated additional functions with evolution of eukaryotes from simple prokaryotes. In mammalian cells, they play key roles in controlling DNA synthesis reactions and the reorganization of replicated DNA at replication forks. Several cell cycle regulation proteins target these processes by affecting PCNA actions
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PMID:PCNA binding proteins. 1057 96


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