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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)

A 48-kDa protein from the budding yeast Saccharomyces cerevisiae is antigenically and structurally similar to S-antigen from retina. Eight anti-S-antigen monoclonal antibodies, directed against distinct epitopes, cross-reacted with a yeast 48-kDa protein. Structural similarity between the bovine and yeast proteins was further demonstrated by comparison of tryptic peptide fragments containing one of these epitopes. This 48-kDa yeast protein appears to be a component of the replicative complex of the cell. It was found associated with immunoaffinity-purified yeast DNA polymerase I-primase and with yeast DNA-replicative complex. The 48-kDa protein was phosphorylated by a protein kinase activity endogenous to the replicative complex preparation. This phosphorylation was dependent on the cell division cycle gene CDC7. In addition, authentic bovine S-antigen, when added to yeast DNA polymerase I-primase, stimulated polymerase activity. These findings suggest that the yeast S-antigen-like protein may play a role in replication, and they raise the possibility that it may be involved in traversal of the G1/S boundary of the cell cycle.
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PMID:A 48-kDa, S-antigen-like phosphoprotein in yeast DNA-replicative complex preparations. 186 Aug 66

The yeast DNA polymerase alpha-primase B subunit functions in initiation of DNA replication. This protein is present in two forms, of 86 and 91 kDa, and the p91 polypeptide results from cell cycle-regulated phosphorylation of p86. The B subunit present in G1 arises by dephosphorylation of p91 while cells are exiting from mitosis, becomes phosphorylated in early S phase, and is competent and sufficient to initiate DNA replication. The B subunit transiently synthesized as a consequence of periodic transcription of the POL12 gene is phosphorylated no earlier than G2. Phosphorylation of the B subunit does not require execution of the CDC7-dependent step and ongoing DNA synthesis. We suggest that posttranslational modifications of the B subunit might modulate the role of DNA polymerase alpha-primase in DNA replication.
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PMID:Cell cycle-dependent phosphorylation and dephosphorylation of the yeast DNA polymerase alpha-primase B subunit. 782 54

Eukaryotic cells coordinate chromosome duplication by assembly of protein complexes at origins of DNA replication and by activation of cyclin-dependent kinase and Cdc7p-Dbf4p kinase. We show in Saccharomyces cerevisiae that although Cdc7p levels are constant during the cell division cycle, Dbf4p and Cdc7p-Dbf4p kinase activity fluctuate. Dbf4p binds to chromatin near the G(1)/S-phase boundary well after binding of the minichromosome maintenance (Mcm) proteins, and it is stabilized at the non-permissive temperature in mutants of the anaphase-promoting complex, suggesting that Dbf4p is targeted for destruction by ubiquitin-mediated proteolysis. Arresting cells with hydroxyurea (HU) or with mutations in genes encoding DNA replication proteins results in a more stable, hyper-phosphorylated form of Dbf4p and an attenuated kinase activity. The Dbf4p phosphorylation in response to HU is RAD53 dependent. This suggests that an S-phase checkpoint function regulates Cdc7p-Dbf4p kinase activity. Cdc7p may also play a role in adapting from the checkpoint response since deletion of CDC7 results in HU hypersensitivity. Recombinant Cdc7p-Dbf4p kinase was purified and both subunits were autophosphorylated. Cdc7p-Dbf4p efficiently phosphorylates several proteins that are required for the initiation of DNA replication, including five of the six Mcm proteins and the p180 subunit of DNA polymerase alpha-primase.
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PMID:Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway. 1050 66

In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes "replicative stress" that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair.
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PMID:Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes. 2995 28

Mcm2-7 is the molecular motor of eukaryotic replicative helicase, and the regulation of this complex is a major focus of cellular S-phase regulation. Despite its cellular importance, few small-molecule inhibitors of this complex are known. Based upon our genetic analysis of synthetic growth defects between mcm alleles and a range of other alleles, we have developed a high-throughput screening (HTS) assay using a well-characterized mcm mutant (containing the mcm2DENQ allele) to identify small molecules that replicate such synthetic growth defects. During assay development, we found that aphidicolin (inhibitor of DNA polymerase alpha) and XL413 (inhibitor of the DNA replication-dependent kinase CDC7) preferentially inhibited growth of the mcm2DENQ strain relative to the wild-type parental strain. However, as both strains demonstrated some degree of growth inhibition with these compounds, small and variable assay windows can result. To increase assay sensitivity and reproducibility, we developed a strategy combining the analysis of cell growth kinetics with linear discriminant analysis (LDA). We found that LDA greatly improved assay performance and captured a greater range of synthetic growth inhibition phenotypes, yielding a versatile analysis platform conforming to HTS requirements.
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PMID:A High-Throughput Assay for DNA Replication Inhibitors Based upon Multivariate Analysis of Yeast Growth Kinetics. 3080 12