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)

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.
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PMID:Reconstitution of simian virus 40 DNA replication with purified proteins. 217 60

The DNA polymerase beta gene (POLB), which encodes a DNA polymerase believed to be involved in short gap-filling DNA synthesis, has been mapped to the proximal region of 8p (8p12-p11), a region commonly deleted in bladder carcinoma and a wide variety of other neoplasms. Also mapped to this region (8p12-p11.2) is the gene encoding the beta isoform of the catalytic subunit of protein phosphatase 2A (PPP2CB), a major serine/threonine phosphatase thought to play a regulatory role in many cellular pathways. The known functions of these proteins make them good candidates for 8p tumor suppressor genes. To test this hypothesis, we assessed a series of bladder tumors and bladder tumor cell lines for sequence variation in POLB and PPP2CB. Single strand conformation polymorphism (SSCP) analysis and direct sequencing of POLB cDNA derived from cell lines and tumors, many with known deletions of proximal 8p, revealed one sequence variant that was shown to represent a normal sequence polymorphism. No tumor-specific sequence variants were identified. The promotor sequence in genomic DNA from tumors with 8p LOH was also screened by SSCP. Four polymorphisms were identified but no tumor-specific mutations were found. PPP2CB was analyzed by SSCP analysis of all 7 coding exons in genomic DNA of bladder tumors and cell lines. Polymorphisms were detected in exons 4 and 5 but no tumor-specific mutations were found. We conclude that these genes are unlikely to be the suppressor genes for bladder cancer targeted by deletions of chromosome arm 8p.
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PMID:Mutation analysis of 8p genes POLB and PPP2CB in bladder cancer. 907 3

Human retinoblastoma (Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10-100-fold the activity of DNA polymerase alpha from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and under-phosphorylated forms. Dephosphorylation of Rb protein by protein phosphatase 2A largely diminished its stimulatory effect. On the other hand, a hyperphosphorylated Rb protein, obtained from insect cells overexpressing Rb protein, cyclin E and cyclin-dependent kinase 2 simultaneously, stimulated DNA polymerase alpha more strongly than the singly-expressed Rb protein. These results indicate that the phosphorylation is crucial for the stimulation. Rb protein isolated from human Burkitt lymphoma Raji cells also stimulated DNA polymerase alpha. In contrast, Rb protein did not affect eukaryotic DNA primase or Klenow fragment of Escherichia coli DNA polymerase I. By immunoprecipitation using anti-DNA polymerase alpha antibody, Rb protein in nuclear extract of Raji cells was co-precipitated with DNA polymerase alpha. This result indicates that DNA polymerase alpha exists as a complex containing phosphorylated Rb protein in cells. DNA polymerase alpha specifically bound to a purified Rb protein-immobilized Sepharose column. Rb protein also bound to DNA polymerase alpha trapped to anti-DNA polymerase alpha antibody-Sepharose column, suggesting the direct association of these two proteins. These observations suggest a new function of phosphorylated Rb protein in the regulation of DNA replication.
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PMID:Phosphorylated retinoblastoma protein stimulates DNA polymerase alpha. 939 44

In eukaryotic cells, an ordered sequence of events leads to the initiation of DNA replication. During the G(1) phase of the cell cycle, a prereplication complex (pre-RC) consisting of ORC, Cdc6, Cdt1, and MCM2-7 is established at replication origins on the chromatin. At the G(1)/S transition, MCM10 and the protein kinases Cdc7-Dbf4 and Cdk2-cyclin E cooperate to recruit Cdc45 to the pre-RC, followed by origin unwinding, RPA binding, and recruitment of DNA polymerases. Using the soluble DNA replication system derived from Xenopus eggs, we demonstrate that immunodepletion of protein phosphatase 2A (PP2A) from egg extracts and inhibition of PP2A activity by okadaic acid abolish loading of Cdc45 to the pre-RC. Consistent with a defect in Cdc45 loading, origin unwinding and the loading of RPA and DNA polymerase alpha are also inhibited. Inhibition of PP2A has no effect on MCM10 loading and on Cdc7-Dbf4 or Cdk2 activity. The substrate of PP2A is neither a component of the pre-RC nor Cdc45. Instead, our data suggest that PP2A functions by dephosphorylating and activating a soluble factor that is required to recruit Cdc45 to the pre-RC. Furthermore, PP2A appears to counteract an unknown inhibitory kinase that phosphorylates and inactivates the same factor. Thus, the initiation of eukaryotic DNA replication is regulated at the level of Cdc45 loading by a combination of stimulatory and inhibitory phosphorylation events.
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PMID:Protein phosphatase 2A regulates binding of Cdc45 to the prereplication complex. 1218 86