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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)

DNA single-strand breaks are caused by aqueous extracts of cigarette tar, due to the reduction of oxygen to superoxide by tar and the subsequent production of hydroxyl radicals. The action of DNA metabolism enzymes on these single-strand breaks has been studied to probe the consequences of these lesions for DNA repair. Our results demonstrate that cigarette tar-induced nicks are blocked at the 3' terminus since they are totally incapable of activating DNA for DNA synthesis by Escherichia coli DNA polymerase I. The 3' termini of these tar-induced nicks are activated, however, for DNA synthesis by E. coli exonuclease III or by the 3' phosphatase activity of T4 polynucleotide kinase. Because of the inability of tar-induced lesions to support DNA synthesis, they probably require a multi-step process for repair in vivo. As a consequence, the overall likelihood of mutation is increased due to the possibility for error at each step of the repair process.
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PMID:DNA synthesis is blocked by cigarette tar-induced DNA single-strand breaks. 282 Jun 3

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

Using dodecadeoxynucleotides as primers for DNA synthesis and 3'-o-chlorophenyl-phosphorylated dodecadeoxynucleotides as "stoppers" for chain elongation, pre-defined regions of a gene previously cloned in M13 single-stranded (ss) DNA phage were converted into double-stranded (ds) DNA utilizing the action of the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). The resulting ds DNA was freed from the ss region by S1 nuclease treatment. This method can be used to obtain DNA fragments of any size with pre-defined 5' and 3' ends. About 15% of the input ss DNA template molecules are converted into ds DNA fragments. This technique was used to synthesize several DNA fragments from different portions of the hepatitis B virus surface antigen (HBsAg) gene. The products were then ligated into a yeast plasmid vector that carries the E. coli lacZ gene which is located downstream from the yeast acid-phosphatase promotor. Using this system, several fragments of HBsAg were produced in the form of beta-galactosidase fused protein.
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PMID:Cloning a defined region of DNA using a limited action of DNA polymerase: application to dissection of hepatitis B virus surface antigen gene. 378 Dec 47

In addition to the previously described deoxyribonucleic acid (DNA) polymerase, DNA ligase, DNA exonuclease, and DNA endonuclease activities, purified virions of Schmidt-Ruppin strain of Rous sarcoma virus (SRV) have nucleotides and nucleotide kinase, phosphatase, hexokinase, and lactate dehydrogenase activities. The SRV virions have no glucose-6-phosphate dehydrogenase activity. All enzyme activities, but glucose-6-phosphate dehydrogenase and adenosine triphosphatase, were increased by disruption of the virions. The DNA polymerase, DNA ligase, and hexokinase activities had a higher specific activity in purified virion cores. It is suggested that during assembly virions of SRV may pick up cytoplasmic components which bind to virion proteins. The role of these components in viral replication is not known at present.
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PMID:Enzymes and nucleotides in virions of Rous sarcoma virus. 433 49

1. The incorporation of thymidine into DNA of regenerating rat liver was measured at various times after partial hepatectomy. A single intravenous injection of 30mumol of beryllium/kg given immediately after the operation inhibited DNA synthesis 12, 16, 20, 24 and 28h later. 2. The activity of several enzymes critical to DNA synthesis (thymidine kinase, thymidylate kinase, thymidylate synthetase, deoxycytidylate deaminase and DNA polymerase) increased in control rats 20-24h after partial hepatectomy severalfold over the activity found in resting livers. After beryllium treatment this rise in activity was much less and it seemed as if beryllium would partially block the induction of DNA-synthesizing enzymes after partial hepatectomy. 3. Enzymes whose activities do not rise during liver regeneration were not affected by beryllium (aspartate transcarbamoylase, carbamoyl phosphate synthetase, uridine kinase and glucose 6-phosphatase). 4. No evidence was found in vitro that beryllium would specifically inhibit thymidine kinase or DNA polymerase. 5. The time-effect relationship between beryllium administration and thymidine kinase activity in vivo was examined. Measured 24h after partial hepatectomy, thymidine kinase activity was only affected if beryllium was given within the first 9-12h after partial hepatectomy. Beryllium given later, even in greatly increased doses, failed to have any effect on thymidine kinase. The possibility is discussed that beryllium inhibits enzyme induction at the transcriptional level.
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PMID:Effects of beryllium on deoxyribonucleic acid-synthesizing enzymes in regenerating rat liver. 549 75

phi X174 RFI DNA treated with bleomycin (BLM) under conditions permitting nicking does not serve as a template-primer for Escherichia coli DNA polymerase I. Purified exonuclease III from E. coli and extracts from wild-type E. coli strains are able to convert the BLM-treated DNA to suitable template-primer, but extracts from exonuclease III deficient strains are not. Brief digestion by exonuclease III is enough to create the template-primer, suggesting that the exonuclease III is converting the BLM-treated DNA by a modification of 3' termini. The exonucleolytic rather than the phosphatase activity of exonuclease III appears to be involved in the conversion. Comparative studies with micrococcal nuclease indicate that BLM-created nicks do not have a simple 3'-P structure. Bacterial alkaline phosphatase does not convert BLM-treated DNA to template-primer. The endonuclease VI activity associated with exonuclease III does not incise DNA treated with BLM under conditions not allowing nicking, in contrast to DNA with apurinic sites made by acid treatment, arguing that conversion does not require the endonuclease VI action on uncleaved sites.
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PMID:Synthesis by DNA polymerase I on bleomycin-treated deoxyribonucleic acid: a requirement for exonuclease III. 616 81

Human placental extracts contain a specific inhibitor of mammalian retroviral RNA-directed DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) activity. This inhibitor copurifies with retrovirus-like particles in human placental extracts. The inhibitor can be removed from these particles by salt extraction, which leads to the recovery of the polymerase activity. Thus, the inhibitor does not irreversibly inactivate the particle-associated RNA-directed DNA polymerase activity. The inhibitory preparation contained no nuclease, protease, or phosphatase activity. Because its inhibitory action can be eliminated by the addition of more virus to the reaction, nonspecific inactivation of enzyme substrate has been ruled out. A partial characterization of the inhibitor indicates that it is (i) insensitive to ether, trypsin, and phospholipase C; (ii) stable to heat and pH 2-12; and (iii) nondialyzable.
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PMID:Human placentas contain a specific inhibitor of RNA-directed DNA polymerase. 616 15

Neocarzinostatin (NCS) induces repair in a xeroderma pigmentosum lymphoblastoid line deficient in the ability to repair DNA damage induced with (acetoxyacetyl-amino)fluorene. Repair was demonstrated by the induction of repair synthesis and by the disappearance of NCS-induced single-strand breaks and/or alkaline-labile sites in DNA. Estimation of NCS-induced repair patch size, based on the density shift induced in DNA by extensive shear after incubation of treated cells in medium with bromodeoxyuridine or by calculation from the extent of restoration of DNA sedimentation profiles in alkaline sucrose gradients and the amount of repair synthesis measured by the BND cellulose method, indicated that only a few nucleotides were inserted per repaired region. NCS-treated bacteriophage T7 DNA requires incubation with alkaline phosphatase to make it a substrate for DNA polymerase I. NCS-reacted T7 DNA, even after phosphatase treatment, is not a substrate for a DNA polymerase alpha obtained from human lymphoma cells. NCS-treated T7 DNA did serve as a substrate for the DNA polymerase alpha when incubated with an apurinic/apyrimidinic (AP) endonuclease with associated 5'-3'-exonuclease activity. The results suggest that NCS-induced AP sites could be intermediates for the in vivo repair synthesis.
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PMID:Repair of neocarzinostatin-induced deoxyribonucleic acid damage in human lymphoblastoid cells: possible involvement of apurinic/apyrimidinic sites as intermediates. 625 59

A protein factor which stimulates DNA polymerase alpha activity on heat-denatured DNA has been purified from mouse FM3A cells. The final preparation had a specific activity of 43,000 units/mg protein and lacked detectable DNA polymerase, RNA polymerase, DNA-dependent- and independent ATPase, exo- and endodeoxyribonuclease and phosphatase activities. The stimulating factor sedimented at 2.9S in a glycerol gradient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the glycerol gradient fraction revealed the presence of a major band of 36,000 daltons, the amount of which corresponded well with the level of stimulating activity. The stimulation by the factor was specific for heat-denatured DNA, and a little or no stimulation was observed with native DNA, ribo- and deoxyribohomopolymers and single stranded circular DNA. Alkaline sucrose gradient sedimentation analysis of the reaction products revealed that newly synthesized DNA was covalently linked to the termini of heat-denatured DNA. The average chain length of the elongated span determined by the digestion with micrococcal nuclease and phosphodiesterase II, did not differ between in the presence and absence of the stimulating factor, suggesting that the stimulation by the factor was due to the increase in the initiation frequency of DNA synthesis from the 3'-hydroxyl terminus of heat-denatured DNA.
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PMID:Purification and characterization of a factor stimulating DNA polymerase alpha activity from mouse FM3A cells. 632 2

There are more than twenty appreciably different deoxyribonucleoside triphosphate assays using DNA polymerase in the literature. Therefore, each aspect of this method has been critically evaluated, including the purity of the substrates and of DNA polymerase, the reaction conditions, product isolation, and the effect of cell extracts on the assay. The final method uses a phosphatase-free DNA polymerase preparation, 2'-dAMP to inhibit the 3' leads to 5' exonuclease of DNA polymerase I, and includes a correction of both the background incorporation and the sample incorporation for dilution of the specific activity of the radioactive precursor by the sample. The sensitivity, range, accuracy, and reproducibility are reported as well as values for the deoxyribonucleoside triphosphate content of Chinese hamster ovary K-1 cells.
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PMID:Determination of deoxyribonucleoside triphosphates using DNA polymerase: a critical evaluation. 731 18


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