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 papovavirus SV40 is able to induce tumours in susceptible hosts and will transform cells in vitro. Its major early protein, large T antigen, is required for viral DNA synthesis, both in vivo and in vitro, and is also responsible for the oncogenic action of the virus. We have made use of an extensive library of anti-T monoclonal antibodies to investigate the cellular effects of T. Large T shares an antigenic determinant with a growth-regulated host protein, p68, which is a member of an expanding super-family of helicases with particular homology to the translation initiation factor elF-4A. We have also studied the binding and interaction of large T with two particular host components: the replicative enzyme DNA polymerase alpha and the proto-oncogene p53. These two proteins bind to similar regions of T and exert similar effects on its antigenic structure. We found that p53 can block the binding of DNA polymerase alpha to T as well as co-existing with DNA polymerase alpha in a trimeric complex with T. This suggests that these interactions may be important in the oncogenic and replicative action of large T.
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PMID:Host proteins that bind to or mimic SV40 large T antigen: using antibodies to look at protein interactions and their significance. 247 59

Recent studies with crude or partially purified cell extracts have suggested that DNA polymerase alpha activity may be regulated by enzymatic phosphorylation. To further investigate these findings, we have examined the effects of protein kinases and phosphatases on highly purified DNA polymerase alpha from mouse cells. Incubation of DNA polymerase alpha with a variety of protein kinases, including protein kinase C, had no effect on polymerase activity. In addition, treatment of the polymerase with soluble calf intestinal alkaline phosphatase had no effect on DNA polymerase alpha activity, further indicating that phosphorylation does not have a direct role in modulating polymerase activity. In contrast, incubation of DNA polymerase alpha with calf intestinal alkaline phosphatase crosslinked to agarose beads resulted in a time dependent disappearance of polymerase activity. This loss of DNA polymerase activity was dependent on phosphatase activity, as the alkaline phosphatase inhibitors, potassium phosphate or levamisole, prevented the loss of polymerase activity in the presence of the beaded phosphatase. The loss of DNA polymerase alpha activity following beaded phosphatase treatment was not a general phenomena as the large fragment of Escherichia coli DNA polymerase I, T4 DNA polymerase or mouse primase were not affected by similar treatment. The decreased DNA polymerase activity following incubation with phosphatase beads correlated with the binding of the DNA polymerase polypeptides, p185 and p68, to the agarose beads and this binding could not be reversed by either 150 mM potassium chloride or sodium sulfate. The binding of the polymerase to the agarose beads was dependent on the phosphatase activity, as the polymerase could be first treated with soluble calf intestinal phosphatase and subsequently bound to added Sepharose 4B beads. Surprisingly, Sepharose CL4B, a highly desulfated agarose preparation, did not bind the phosphatase-treated polymerase suggesting that sulfated polysaccharides are required for polymerase binding. The physiological correlate of this binding is unknown, but it has been reported that sulfated polysaccharides exist in a variety of intracellular compartments. It would be interesting to speculate that phosphorylation controls the intracellular compartmentalization of DNA polymerase alpha.
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PMID:DNA polymerase alpha activity is not affected by protein kinases or alkaline phosphatase. 293 May 69

Mouse cell extracts support vigorous replication of polyomavirus (Py) DNA in vitro, while human cell extracts do not. However, the addition of purified mouse DNA polymerase alpha-primase to human cell extracts renders them permissive for Py DNA replication, suggesting that mouse polymerase alpha-primase determines the species specificity of Py DNA replication. We set out to identify the subunit of mouse polymerase alpha-primase that mediates this species specificity. To this end, we cloned and expressed cDNAs encoding all four subunits of mouse and human polymerase alpha-primase. Purified recombinant mouse polymerase alpha-primase and a hybrid DNA polymerase alpha-primase complex composed of human subunits p180 and p68 and mouse subunits p58 and p48 supported Py DNA replication in human cell extracts depleted of polymerase alpha-primase, suggesting that the primase heterodimer or one of its subunits controls host specificity. To determine whether both mouse primase subunits were required, recombinant hybrid polymerase alpha-primases containing only one mouse primase subunit, p48 or p58, together with three human subunits, were assayed for Py replication activity. Only the hybrid containing mouse p48 efficiently replicated Py DNA in depleted human cell extracts. Moreover, in a purified initiation assay containing Py T antigen, replication protein A (RP-A) and topoisomerase I, only the hybrid polymerase alpha-primase containing the mouse p48 subunit initiated primer synthesis on Py origin DNA. Together, these results indicate that the p48 subunit is primarily responsible for the species specificity of Py DNA replication in vitro. Specific physical association of Py T antigen with purified recombinant DNA polymerase alpha-primase, mouse DNA primase heterodimer, and mouse p48 suggested that direct interactions between Py T antigen and primase could play a role in species-specific initiation of Py replication.
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PMID:The mouse DNA polymerase alpha-primase subunit p48 mediates species-specific replication of polyomavirus DNA in vitro. 786 63

DNA-polymerase-alpha--primase complex contains four subunits, p180, p68, p58, and p48, and comprises a minimum of two enzymic functions. We have cloned cDNAs encoding subunits of DNA-polymerase-alpha--primase from human and mouse. Sequence comparisons showed high amino acid conservation among the mammalian proteins. We have over-expressed the single polypeptides and co-expressed various subunit complexes using baculovirus vectors, purified the proteins and investigated their biochemical properties. The purified mouse p48 subunit (Mp48) alone had primase activity. Purification of co-expressed Mp48 and Mp58 subunits yielded stable DNA primase of high specific activity. Co-expression of all four subunits yielded large quantities of tetrameric DNA-polymerase-alpha--primase. The p180, p58 and p48 polypeptides were also co-expressed and immunoaffinity purified as a trimeric enzyme complex. The tetrameric and trimeric DNA-polymerase-alpha--primase complexes showed both DNA primase and DNA polymerase activities. The tetrameric recombinant DNA-polymerase-alpha--primase synthesized double-stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.
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PMID:DNA replication in vitro by recombinant DNA-polymerase-alpha-primase. 802 92

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alpha-primase was deleted, while MMMM and the MMHH complex did not. To determine whether the human p180 or p68 subunit was required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HMMM and MHMM) or three human subunits and one mouse subunit (MHHH and HMHH) were tested for SV40 DNA replication activity. The hybrid complexes HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiation assay with purified proteins and replicated SV40 DNA in depleted mouse extracts. In contrast, the hybrid complexes containing mouse p180 were inactive in both assays. We conclude that the human p180 subunit determines host-specific replication of SV40 DNA in vitro.
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PMID:Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase alpha-primase. 852 33

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

DNA polymerase alpha-primase is a replication enzyme necessary for DNA replication in all eukaryotes examined so far. Mouse DNA polymerase alpha is made up of four subunits, the largest of which is the catalytic subunit with a molecular mass of 180 kDa (p180). This subunit exists as a tight complex with the second-largest subunit (p68), whose physiological role has remained unclear up until now. We set out to characterize these subunits individually or in combination by using a cDNA expression system in cultured mammalian cells. Coexpression of p68 markedly increased the protein level of p180, with the result that ectopically generated DNA polymerase activity was dramatically increased. Immunofluorescence analysis showed that while either singly expressed p180 or p68 was localized in the cytoplasm, cotransfection of both subunits resulted in colocalization in the nucleus. We identified a putative nuclear localization signal for p180 (residues 1419 to 1437) and found that interaction with p68 is essential for p180 to translocate into the nucleus. These results indicate that association of p180 with p68 is important for both protein synthesis of p180 and translocation into the nucleus, implying that p68 plays a pivotal role in the newly synthesized DNA polymerase alpha complex.
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PMID:The second-largest subunit of the mouse DNA polymerase alpha-primase complex facilitates both production and nuclear translocation of the catalytic subunit of DNA polymerase alpha. 958 95

The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase alpha-primase (polalpha-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polalpha-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind the ori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polalpha-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.
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PMID:A C-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit. 969 37

DNA polymerase alpha-primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions. To study the primase activity of the complex, we expressed cDNAs encoding for the human p58 and p48 subunits either as single proteins or together using Escherichia coli expression vectors. Co-expression of both primase subunits allowed the purification of a heterodimer in high yields that revealed stable primase activity. Purified recombinant p48 subunit showed enzyme activity, whereas purified p58 did not. In contrast to the heterodimer, the primase activity of p48 was unstable. The activity of p48 could be stabilized by the addition of the divalent cations Mg2+ and Mn2+ but not Zn2+. On a poly(dC) template the primase activity was hardly influenced by the monovalent cation potassium. However, by using poly(dT) as a template the recombinant p48 activity was sensitive to salt, whereas recombinant p58-p48 and the bovine DNA polymerase alpha-primase purified from thymus were less sensitive to the addition of monovalent cations. A complex of bacterially expressed primase and baculovirus-expressed p180 and p68 was assembled in vitro and shown to support replication of simian virus 40 DNA in a cell-free system.
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PMID:Primase activity of human DNA polymerase alpha-primase. Divalent cations stabilize the enzyme activity of the p48 subunit. 970 92

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


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