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)

Uracil-DNA glycosylase from rat liver mitochondria, an inner membrane protein, has been purified approximately 575,000-fold to apparent homogeneity. During purification two distinct activity peaks, designated form I and form II, were resolved by phosphocellulose chromatography. Form I constituted approximately 85% while form II was approximately 15% of the total activity; no interconversion between the forms was observed. The major form was purified as a basic protein with an isoelectric point of 10.3. This enzyme consists of a single polypeptide with an apparent Mr of 24,000 as determined by recovering glycosylase activity from a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 29,000 was determined by glycerol gradient sedimentation. The purified enzyme had no detectable exonuclease, apurinic/apyrimidinic endonuclease, DNA polymerase, or hydroxymethyluracil-DNA glycosylase activity. A 2-fold preference for single-stranded uracil-DNA over a duplex substrate was observed. The apparent Km for uracil residues in DNA was 1.1 microM, and the turnover number is about 1000 uracil residues released per minute. Both free uracil and apyrimidinic sites inhibited glycosylase activity with Ki values of approximately 600 microM and 1.2 microM, respectively. Other uracil analogues including 5-(hydroxymethyl)uracil, 5-fluorouracil, 5-aminouracil, 6-azauracil, and 2-thiouracil or analogues of apyrimidinic sites such as deoxyribose and deoxyribose 5'-phosphate did not inhibit activity. Both form I and form II had virtually identical kinetic properties, and the catalytic fingerprints (specificity for uracil residues located in a defined nucleotide sequence) obtained on a 152-nucleotide restriction fragment of M13mp2 uracil-DNA were almost identical. These properties differentiated the mitochondrial enzyme from that of the uracil-DNA glycosylase purified from nuclei of the same source.
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PMID:Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver. 319 81

The activities of the DNA repair enzymes O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase, and the replicative enzyme DNA polymerase alpha, were measured in extracts of human fetal tissues at 18-20 weeks of gestation. In general, O6-methylguanine-DNA methyltransferase activities in fetal tissues were in the same range as in the corresponding adult tissues, except for fetal liver which had approximately 5-fold lower activity. Uracil-DNA glycosylase was, surprisingly, approximately 4-fold lower in fetal tissues compared with adult tissues. Since a critical factor in carcinogenesis may be the rate of repair relative to DNA replication, the activities of O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase were compared with the DNA polymerase alpha activity in the same extract. When expressed in this way, O6-methylguanine-DNA methyltransferase activity was lowest in liver and brain and 2- to 14-fold higher in kidney, lung, colon, stomach, small intestine and pancreas. The ratio of uracil-DNA glycosylase to DNA polymerase alpha varied less between different organs. These findings indicate that several fetal organs may be more sensitive than adult organs to some alkylating agents that are known to occur in the environment. Furthermore, the lower capacity of DNA repair is not restricted to repair of alkylation damage, since the activity of uracil-DNA glycosylase is also lower than in adult tissues.
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PMID:Repair of premutagenic DNA lesions in human fetal tissues: evidence for low levels of O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase activity in some tissues. 665 68

Heat treatment of poly(deoxycytidylic acid)-[poly(dC)] induces the formation of dUMP residues, which code for dAMP when replicated by Escherichia coli DNA polymerases I and III. The specificity of dUMP coding properties is indicated by the quantitative relation between the dAMP incorporated and the frequency of dUMP residues in the heat-treated poly(dC). The dAMP incorporation is prevented by preincubation of uracil containing poly(dC) with uracil-DNA glycosylase. The excision of uracil by uracil-DNA glycosylase leads to the formation of apyrimidinic sites (AP sites), which are barely replicated in vitro under physiological conditions. However, the alteration of E. coli DNA polymerase I fidelity of replication by Mn2+ greatly stimulates the replication of AP sites. There is a preferential incorporation of dAMP, as compared to dTMP, opposite the AP sites. The dAMP incorporation is prevented by preincubation of poly(dC) containing AP sites with Micrococcus luteus AP endonuclease B. The results show a close association between DNA repair by base excision and the prevention of mutagenic processes in vitro. Furthermore, since the alteration of DNA polymerase fidelity allows some replication of the noncoding DNA lesion (AP site), this could imply a role in SOS-induced mutagenesis in vivo.
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PMID:Coding properties of poly(deoxycytidylic acid) templates containing uracil or apyrimidinic sites: in vitro modulation of mutagenesis by deoxyribonucleic acid repair enzymes. 676 Aug 93

Plasmids were constructed with guanine (G) or O6-methyl- (m6G), O6-ethyl-(e6G), or O6-benzyl- (b6G) guanine in the initiation codon (ATG) of the lacZ' gene. Four deoxyuridine residues were incorporated near the modified guanine in the complementary strand. The deoxyuridine-containing plasmids exhibited similarly high transformation efficiencies in ung- Escherichia coli, although the frequency of mutations induced by m6G, e6G, and b6G residues was relatively low. Treatment of the plasmids with uracil-DNA glycosylase (UDG), to remove the uracil residues, or UDG and exonuclease III, to create a gap in the deoxyuridine-containing strand, reduced transformation efficiency for adduct-containing plasmids but did not affect transformation efficiency for control plasmids. However, the same treatments dramatically enhanced mutagenesis by m6G, e6G, and b6G. These results were consistent with blockage of replication by the modified guanines in double-stranded plasmids resulting in preferential replication of the complementary strand. Replication past the modified guanines was forced in the gapped plasmids. The frequency of modified guanine-induced mutations in gapped vectors was similar in strains of E. coli that were proficient in DNA polymerase III but deficient in either DNA polymerase I or II or both polymerase I and II suggesting either that polymerase III was primarily responsible for adduct bypass in all strains or that the probability of base misinsertion during bypass by either polymerase I or II was similar to that for polymerase III. Repair studies with gapped plasmids indicated that m6G was subject to repair by Ada methyltransferase and to postreplication processing by methylation-directed mismatch repair. Neither e6G nor b6G were similarly repaired.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mutagenesis in Escherichia coli by three O6-substituted guanines in double-stranded or gapped plasmids. 761 34

The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.
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PMID:DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract. 782 35

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying DNA that is based upon the combined action of a DNA polymerase and restriction enzyme. Previously, a form of SDA was developed which utilizes the exonuclease deficient Klenow fragment of E. coli polymerase I (exo Klenow) and the restriction enzyme HincII to achieve 10(8)-fold amplification in 2 h at 37 degrees C (Walker, G.T., 1993, PCR Methods and Applications 3; 1-6). A new thermophilic form of SDA is reported here which uses a restriction endonuclease from Bacillus stearothermophilus (BsoBI) and a 5'-->3' exonuclease deficient polymerase from Bacillus caldotenax (exo Bca). SDA was used to amplify DNA from Mycobacterium tuberculosis. An amplification factor of 10(10)-fold was achieved after 15 min of SDA at 60 degrees C. The new thermophilic system is much more specific than the previous mesophilic system as evidenced by a dramatic decrease in background amplification products. Thermophilic SDA was also optimized with dUTP substituted for TTP to enable amplicon decontamination using uracil-DNA glycosylase.
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PMID:Detection of M. tuberculosis DNA using thermophilic strand displacement amplification. 886 73

Repair of a uracil-guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two-hybrid analyses. In addition, a complex formed between DNA polymerase beta and a double-stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay. The region of interaction with DNA polymerase beta is located within residues 84-183 in the N-terminal half of the XRCC1 protein, whereas the C-terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision-repair. DNA strand displacement and excessive gap filling during DNA repair were observed in cell-free extracts of an XRCC1-deficient mutant cell line, in agreement with the results from the reconstituted system.
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PMID:Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein. 897 92

Base excision repair is a major mechanism for correcting aberrant DNA bases. We are using an in vitro base excision repair assay to fractionate and purify proteins from a human cell extract that are involved in this type of repair. Three fractions are required to reconstitute base excision repair synthesis using a uracil-containing DNA as a model substrate. We previously showed that one fraction corresponds to DNA polymerase beta. A second fraction was extensively purified and found to possess uracil-DNA glycosylase activity and was identified as the product of the UNG gene. A neutralizing antibody to the human UNG protein inhibited base excision repair in crude extract by at least 90%. The third fraction was highly purified and exhibited apurinic/apyrimidinic (AP) endonuclease activity. Immunoblot analysis identified HAP1 as the major polypeptide in fractions possessing DNA repair activity. Recombinant versions of UNG, HAP1, and DNA polymerase beta were able to substitute for the proteins purified from human cells. Addition of DNA ligase I led to ligated repair products. Thus, complete base excision repair of uracil-containing DNA was achieved by a combination of UNG, HAP1, DNA polymerase beta, and DNA ligase I. This is the first complete reconstitution of base excision repair using entirely eukaryotic proteins.
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PMID:Reconstitution of human base excision repair with purified proteins. 920 Jul 7

The Herpesviridae comprise a large class of animal viruses of considerable public health importance. Of the Herpesviridae, replication of herpes simplex virustype-1 (HSV-1) has been the most extensively studied. The linear 152-kbp HSV-1 genome contains three origins of DNA replication and approximately 75 open-reading frames. Of these frames, seven encode proteins that are required for originspecific DNA replication. These proteins include a processive heterodimeric DNA polymerase, a single-strand DNA-binding protein, a heterotrimeric primosome with 5'-3' DNA helicase and primase activities, and an origin-binding protein with 3'-5' DNA helicase activity. HSV-1 also encodes a set of enzymes involved in nucleotide metabolism that are not required for viral replication in cultured cells. These enzymes include a deoxyuridine triphosphatase, a ribonucleotide reductase, a thymidine kinase, an alkaline endo-exonuclease, and a uracil-DNA glycosylase. Host enzymes, notably DNA polymerase alpha-primase, DNA ligase I, and topoisomerase II, are probably also required. Following circularization of the linear viral genome, DNA replication very likely proceeds in two phases: an initial phase of theta replication, initiated at one or more of the origins, followed by a rolling-circle mode of replication. The latter generates concatemers that are cleaved and packaged into infectious viral particles. The rolling-circle phase of HSV-1 DNA replication has been reconstituted in vitro by a complex containing several of the HSV-1 encoded DNA replication enzymes. Reconstitution of the theta phase has thus far eluded workers in the field and remains a challenge for the future.
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PMID:Herpes simplex virus DNA replication. 924 11

Uracil can arise in DNA by misincorporation of dUTP into nascent DNA and/or by cytosine deamination in established DNA. Based on recent findings, both pathways appear to be promoted in the methyl-deficient model of hepatocarcinogenesis. A chronic increase in the ratio dUTP:dTTP with folate/methyl deficiency can result in a futile cycle of excision and reiterative uracil misincorporation leading to premutagenic apyrimidinic (AP) sites, DNA strand breaks, DNA fragmentation and apoptotic cell death. The progressive accumulation of unmethylated cytosines with chronic methyl deficiency will increase the potential for cytosine deamination to uracil and further stress uracil mismatch repair mechanisms. Uracil is removed by a highly specific uracil-DNA glycosylase (UDG) leaving an AP site that is subsequently repaired by sequential action of AP endonuclease, 5'-phosphodiesterase, a DNA polymerase and DNA ligase. Since the DNA polymerases cannot distinguish between dUTP and dTTP, an increase in dUTP:dTTP ratio will promote uracil misincorporation during both DNA replication and repair synthesis. The misincorporation of uracil for thymine (5-methyluracil) may constitute a genetically significant form of DNA hypomethylation distinct from cytosine hypomethylation. In the present study a significant increase in the level of uracil in liver DNA as early as 3 weeks after initiation of folate/methyl deficiency was accompanied by parallel increases in DNA strand breaks, AP sites and increased levels of AP endonuclease mRNA. In addition, uracil was also detected within the p53 gene sequence using UDG PCR techniques. Increased levels of uracil in DNA implies that the capacity for uracil base excision repair is exceeded with chronic folate/methyl deficiency. It is possible that enzyme-induced extrahelical bases, AP sites and DNA strand breaks interact to negatively affect the stability of the DNA helix and stress the structural limits of permissible uracil base excision repair activity. Thus substitution of uracil for thymine induces repair-related premutagenic lesions and a novel form of DNA hypomethylation that may relate to tumor promotion in the methyl-deficient model of hepatocarcinogenesis.
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PMID:Presence and consequence of uracil in preneoplastic DNA from folate/methyl-deficient rats. 939 4


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