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)

We have investigated the mechanism of bromouracil-induced transition mutations in vitro using synthetic DNA templates and purified T4 DNA polymerase. Evidence is presented for the occurrence of bromouracil-guanine base pairs in product DNA in the G x C----A x T pathway where guanine is present in the DNA template and bromouracil is present as the deoxynucleoside triphosphate substrate 5-bromodeoxyuridine triphosphate. This finding supports a widely known but as yet untested model proposed by Freese (Freese, E. (1959) J. Mol. Biol. 1, 87-105) in which bromouracil-guanine base pairs are intermediates in 5-bromodeoxyuridine-induced transition mutation pathways. We find that the newly formed B x G base pairs are proofread with an efficiency of 75-85% by the 3' -exonuclease of T4 polymerase. The insertion of bromouracil occurring in direct competition with cytosine deoxyribonucleotides opposite template guanine sites is 1.1 +/- 0.14% (mean +/- S.E.), and the misincorporation ratio, inc(B)/inc(C), is reduced 6-fold by the action of the proofreading exonuclease to 0.16 +/- 0.02% (mean +/- S.E.). A previous study by Trautner et al. (Trautner, T. A., Swartz, M. N., and Kornberg, A. (1962) Proc. Natl. Acad. Sci. U. S. A. 48, 449-455) suggested that, while template bromouracil stimulates incorporation of dGMP in the A x T----G x C transition mutation pathway, it may not be occurring exclusively by the pathway proposed by Freese. We concur with these earlier results, and, in addition, we find the surprising result that the 3'-exonuclease activity of wild-type T4 polymerase removes little or no incorporated dGMP on bromouracil-containing templates.
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PMID:The biochemical basis of 5-bromouracil-induced mutagenesis. Heteroduplex base mispairs involving bromouracil in G x C----A x T and A x T----G x C mutational pathways. 608 45

The nascent DNA synthesized by permeable cells of Bacillus subtilis in the presence of 5'-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP has been isolated and characterized. The newly synthesized DNA was isolated free from other cellular nucleic acids by affinity chromatography on thiol-substituted agarose. The number average chain length of the nascent DNA synthesized in one minute at 25 degrees C was 33 nucleotide residues, due to the chain-terminating action of 2',3'-dideoxyATP. Several lines of evidence indicated that at least 90% of the DNA thus isolated carried a terminally phosphorylated RNA moiety at its 5'-end: (1) the nascent DNA was resistant to exonucleolytic degradation by spleen phosphodiesterase unless first hydrolyzed by strong alkali or ribonuclease; (2) the 5'-termini of nascent DNA could not be phosphorylated by polynucleotide kinase unless first treated with alkaline phosphatase or subjected to hydrolysis by strong alkali or ribonuclease; (3) alkaline hydrolysis of nascent DNA labeled with 32P at the 5'-end released unlabeled DNA with a free 5'-terminus and 32P-labeled ribonucleoside 3',5'-bisphosphates; (4) ribonuclease degradation of similarly labeled material produced an unlabeled DNA-containing polynucleotide fraction and 32P-labeled ribo-oligonucleotides; (5) chromatography on dihydroxyboryl cellulose showed that the RNA moiety lacked a 3'-terminal cis-diol grouping (even after treatment with alkaline phosphatase) unless first subjected to the 3'-exonucleolytic action of bacteriophage T4 DNA polymerase. The sequence of the ribonucleotide chains was elucidated by end-group labeling with polynucleotide kinase and digestion with various ribonucleases. The ribonucleotide moiety was primarily three and four residues in length with the predominant sequence (pp)pApG(pC)1-2pDNA. The possibility that it represents a primer for discontinuous DNA synthesis is discussed.
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PMID:Analysis of the 5'-termini of nascent DNA chains synthesized in permeable cells of Bacillus subtilis. 618 36

Apurinic sites were excised from phi X174 RF DNA with two enzymes isolated from rat liver chromatin: an apurinic/apyrimidinic endodeoxyribonuclease and a 5'-3'-exonuclease; the resulting gap was filled with DNA polymerase beta also prepared from rat liver chromatin and the repair was fully terminated with T4 ligase.
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PMID:Excision of apurinic sites from DNA with enzymes isolated from rat-liver chromatin. 621 70

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

Novikoff rat hepatoma and bovine liver DNAs were digested with Msp I or Hpa II. Restriction fragments were end-labeled using [alpha-32P]-dCTP and the Klenow fragment of E. coli DNA polymerase I and then digested to 2'-deoxyribonucleoside-3'-monophosphates using micrococcal nuclease and spleen phosphodiesterase. Mononucleotides were separated by two-dimensional thin layer chromatography, localized by radioautography, and the [32P]-label quantitated by scintillation spectrometry. This method, based on known specificities of Msp I and Hpa II, shows that CCGG, CMGG, and MCGG (M refers to 5-methylcytosine) occur at frequencies of 89.6%, 1.4%, and 9.0%, respectively, in the rat DNA and at 41.6%, 48.3%, and 10.0%, respectively, in the bovine DNA. [32P] recovery in 3'-5-MedCMP from end-labeled Msp I digests was negligible compared to recovery from Hpa II digests. Hence, Msp I is sensitive to methylation at the 5' cytosine in the sequence CCGG.
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PMID:The 5'-cytosine in CCGG1 is methylated in two eukaryotic DNAs and Msp I is sensitive to methylation at this site. 625 19

Treatment with native DNA polymerase I of Escherichia coli with the acylating agent N-carboxymethylisatoic acid anhydride (NCMIA) results under specific conditions in a rapid loss of polymerase activity, an increase in 5' leads to 3'-exonuclease activity and in unchanged 3' leads to 5'-exonuclease activity. When a nucleoside triphosphate and Mg2+ was present the polymerase activity was completely protected against the effect of NCMIA. Treatment with higher concentration of the acylating agent under these conditions led to a loss of 3' leads to 5'-exonuclease activity without any appreciable loss of polymerase activity. Treatment with NCMIA of the two catalytically active fragments of the enzyme led to very similar results. In this case both the polymerase activity and the 3' leads to 5'-exonuclease activity deteriorated more rapidly on treatment with the acylating reagent. The increase in 5' leads to 3'-exonuclease activity as a result of modification of the native enzyme appeared to be due to a change in the optimum conditions with regard to concentration of the assay buffer used. These changes are very similar to those seen when the polymerase is cleaved by limited proteolysis. From the results obtained it is concluded that NCMIA reacts primarily with a site at or near the triphosphate-Mg2+ complex binding site, leading to an almost complete loss of polymerase activity. The acylating reagent reacts also with another group on the native enzyme resulting in a modification of the 5' leads to 3'-exonuclease activity, and at high concentrations with a group leading to a slow loss of 3' leads to 5'-exonuclease activity.
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PMID:Differential effect of N-carboxymethylisatoylation on the DNA polymerase activity, the 5' leads to 3'-exonuclease activity and the 3' leads to 5'-exonuclease activity of DNA polymerase I of Escherichia coli. 626 20

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

A comparison of DNA polymerase III core enzyme (McHenry, C. S., and Crow, W. (1979) J. Biol. Chem. 254, 1748-1753) prepared from wild type Escherichia coli and a strain harboring the mutator gene, mutD5 (Degnen, G. E., and Cox, E. C. (1974) J. Bacteriol. 17, 477-487) has revealed several differences in their properties. Among these are alterations in the heat stability, divalent cation requirement, pH optimum, 3'----5'-single strand exonuclease activity, and DNA-dependent conversion of a deoxynucleoside triphosphate to its corresponding monophosphate ("turnover"). The decrease in the 3'-single strand exonuclease and turnover indicate a defect in the editing function of the mutD strain, which is at least in part responsible for the high spontaneous mutation rate in mutD. Transformation of mutD by a hybrid plasmid, pRD3, constructed from an EcoRI restriction fragment of E. coli and pBR322, cures mutD of its abnormally high mutation rate, and simultaneously restores its 3'-exonuclease activity. These observations are consistent with the notion that the mutD gene product is a subunit of DNA polymerase III, and it either contains the catalytic site for the 3'-exonuclease or modulates its activity. From a consideration of the known molecular weights of the subunits in DNA polymerase III core (McHenry C. S., and Crow, W. (1979) J. Biol. Chem. 254, 1748-1753) the molecular weights of the two proteins translated in maxicells transformed with pRD3, and from a comparison of our results with those obtained with the mutator dnaQ (Horiuchi, T., Maki, H., Maruyama, M., and Sekiguchi, M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3770-3774) and the work of Cox and Horner (Cox, E. C., and Horner, D. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2295-2299) as well as Echols et al. (Echols, H., Lu, C., and Burgers, P. M. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2189-2192) we tentatively assign the mutD gene product to the epsilon subunit of DNA polymerase III.
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PMID:The interaction of DNA polymerase III and the product of the Escherichia coli mutator gene, mutD. 632 41

DNA polymerase has been purified approximately 2000-fold from Mycobacterium tuberculosis H37Rv. The purified preparation was homogeneous by electrophoretic criteria and has a molecular weight of 135 000. The purified enzyme resembles Escherichia coli polymerase I in its properties, being insensitive to sulfhydryl drugs and possessing 5',3'-exonuclease activity in addition to polymerase and 3',5'-exonuclease activities. However, it differs from the latter in its sensitivity to higher salt concentration and DNA intercalating agents such as 8-aminoquinoline. The polymerase exhibited maximal activity between 37--42 degrees C and pH 8.8--9.5. The polymerase was stable for several months below 0 degree C. However, the 5',3'-exonuclease activity was more labile. The effects of different metal ions, polyamines and drugs on the polymerase activity are presented.
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PMID:Purification and properties of DNA polymerase from Mycobacterium tuberculosis H37Rv. 678 93

The 5'-->3'-exonuclease domain of Escherichia coli DNA polymerase I is required for the completion of lagging strand DNA synthesis, and yet this domain is not present in any of the eukaryotic DNA polymerases. Recently, the gene encoding the functional and evolutionary equivalent of this 5'-->3'-exonuclease domain has been identified. It is called FEN-1 in mouse and human cells and RTH1 in Saccharomyces cerevisiae. This 42-kDa enzyme is required for Okazaki fragment processing. Here we report that FEN-1 physically interacts with proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerases delta and epsilon. Through protein-protein interactions, PCNA focuses FEN-1 on branched DNA substrates (flap structures) and on nicked DNA substrates, thereby stimulating its activity 10-50-fold but only if PCNA can functionally assemble as a toroidal trimer around the DNA. This interaction is important in the physical orchestration of lagging strand synthesis and may have implications for how PCNA stimulates other members of the FEN-1 nuclease family in a broad range of DNA metabolic transactions.
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PMID:Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen. 767 86


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