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)

A novel assay to detect strand-specific DNA repair after cellular exposure to cisplatin at IC50 levels, is used to measure rapid repair in the divergent upstream gene (DUG), a human MutS homolog, and in the bidirectional promoter for dihydrofolate reductase gene (DHFR) and the contiguous upstream DUG. Single-stranded DNA capable of hybridizing to gene-specific probes is generated enzymatically by the 3'-5' exonuclease activity of T4 DNA polymerase. The presence of cisplatin lesions inhibit the exonucleolytic activity of T4 DNA polymerase and block the formation of single-stranded DNA. This decreases the amount of complementary sequence produced when assayed by gene-specific probe hybridization. With the progression of repair, increasing quantities of single-stranded DNA become available for probe hybridization. This assay was applied to human A2780 ovarian carcinoma cells treated with cisplatin at the beginning of G1 phase. A dose-response experiment showed that the assay was applicable down to cisplatin concentrations of 2.5 microM. To assay for strand-specific gene repair, the synchronized cells were treated with cisplatin and then allowed time to repair in drug-free medium. Extensive removal of cisplatin lesions after 2 hr of cellular repair during early G1 phase in the DUG and the DUG/DHFR promoter was measured, with no evidence of repair in the unexpressed delta-globin gene. The extent of preferential DNA repair was much more distinct than has been observed previously at high-drug dosage in asynchronous cells.
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PMID:Rapid gene-specific repair of cisplatin lesions at the human DUG/DHFR locus comprising the divergent upstream gene and dihydrofolate reductase gene during early G1 phase of the cell cycle assayed by using the exonucleolytic activity of T4 DNA polymerase. 797 95

Limited proteolysis of T4 DNA polymerase generated a 45-kDa and 35-kDa protein complex, which had 3'-5' exonucleolytic activity but lacked polymerase activity. After partial chymotryptic digestion of the cloned and expressed 45-kDa protein derived from T4 DNA polymerase, we isolated a 27-kDa fragment (residues 96-331) that still had 3'-5' exonuclease activity, thus demonstrating that the amino acid residues required for catalysis are included within this fragment. We also show that the apparent Km values for the 3'-5' exonuclease activity exhibited by the 27-kDa fragment are considerably greater than the apparent Km values determined for the intact DNA polymerase on deoxyoligonucleotide substrates having more than 3 bases. In contrast, the kcat values for phosphodiester bond hydrolysis of 3'-terminal nucleotides are not very different when comparing intact T4 DNA polymerase with the 27-kDa fragment derived from it. Thus, participation of residues distal to 331 are not required for catalysis, but only for binding, and, based on the similarity of kcat values, the geometry of the residues responsible for catalysis are preserved even in the absence of the carboxyl-terminal 567 residues.
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PMID:Isolation, characterization, and kinetic properties of truncated forms of T4 DNA polymerase that exhibit 3'-5' exonuclease activity. 803 91

The 1235 residue herpes simplex virus DNA polymerase is a prototype alpha-like DNA polymerase and also an antiviral drug target. To investigate its organization, we mapped favored cleavage sites for seven proteases and identified three major classes of stable proteolytic fragments: 70-85-kDa N-terminal fragments, 50-70-kDa fragments that start near residues 600-700, and 12-kDa C-terminal fragments. In coimmunoprecipitation experiments, the first two classes of fragments remained associated; thus, cleavage in the center of the protein did not resolve structurally separate domains. In contrast, the 12-kDa C-terminal fragments did not remain associated with other fragments, suggesting a small separable C-terminal domain. The 70-85-kDa N-terminal fragments contained 3'-5' exonuclease and ribonuclease H activities; however, cleavage at the center of the molecule or near the C terminus appeared to destroy DNA polymerase activity. All three major classes of fragments bound DNA in DNA-cellulose chromatography and Southwestern blot analyses. The C-terminal fragments bound the viral polymerase processivity factor, UL42. The results map activities to regions of herpes simplex virus polymerase and suggest a model for its organization that may be pertinent to other DNA polymerases.
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PMID:Structural and functional organization of herpes simplex virus DNA polymerase investigated by limited proteolysis. 807 31

The mosquito homolog of mammalian DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen (PCNA)-independent form of DNA polymerase delta, has been purified from mosquito larval extracts. The polymerase epsilon was separated from DNA polymerase alpha by chromatography on hydroxylapatite, and the enzyme was subsequently purified on single-stranded DNA agarose, followed by a 5' AMP-agarose chromatography step. The purified polymerase exhibits an intrinsic 3'-5' exonuclease activity and shows high activity using an oligo-primed DNA template. Neither human nor Drosophila PCNA stimulated this polymerase activity. Additional immunochemical and biochemical evidence indicates that this enzyme is distinct from DNA polymerase alpha.
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PMID:Mosquito DNA polymerase epsilon. 809 89

DNA secondary and tertiary structures are known to affect the reaction between the double helix and several damaging agents. We have previously shown that the tertiary structure of DNA influences the reactivity of 4-acetoxyaminoquinoline 1-oxide (Ac-4-HAQO), the ultimate carcinogen of 4-nitroquinoline 1-oxide (4-NQO), being more reactive with naturally supercoiled DNA than with relaxed DNA. The relative proportion of the three main stable adducts and of an unstable adduct, that resulted in strand scission and/or AP sites, was also affected by the degree of supercoiling of plasmid DNA. In this study we examined the influence of Z-DNA structure on the reactivity of Ac-4-HAQO by mapping the distribution of the two main Ac-4-HAQO adducts, C8-guanine and N2-guanine, along a (dC-dG)16 sequence inserted at the BamHI site of pBR322 plasmid DNA. This insert adopted the left-handed Z and right-handed B structure depending on the superhelical density of the plasmid. Sites of C8-guanine adduct formation were determined by hot piperidine cleavage of Ac-4-HAQO modified DNA, while N2-guanine adducts were mapped by the arrest of the 3'-5' exonuclease activity of T4 DNA polymerase. The results showed that Ac-4-HAQO did not react with guanine residues when the (dC-dG)16 sequence was in Z conformation, while hyperreactivity at the B-Z junction was observed. These results indicate that Ac-4-HAQO can probe the polymorphism of DNA at the nucleotide level.
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PMID:The ultimate carcinogen of 4-nitroquinoline 1-oxide does not react with Z-DNA and hyperreacts with B-Z junctions. 812 67

In this study we report on the evidence that an alpha-like DNA polymerase purified from the thermoacidophilic archaeon Sulfolobus solfataricus has a modular organization of its associated catalytic activities (polymerase and 3'-5' exonuclease). This enzyme, a monomer of about 100 kDa whose complete primary structure is available, has a protease hypersensitive site that is likely to be cleaved by the action of endogenous proteases during the purification procedure. As a consequence of that, two proteolytic fragments of about 50 and 40 kDa, in addition to the intact 100-kDa molecular species, can be detected upon SDS-PAGE of highly purified S. solfataricus DNA polymerase samples. The amino-terminal microsequence analysis by Edman degradation has revealed that the 50- and the 40-kDa polypeptides correspond to the carboxyl- and the amino-terminal portion of the protein molecule, respectively. Using the bidimensional activity gel assay procedure, recently described by Longley and Mosbaugh (Longley, M. J., and Mosbaugh, D. W. (1991) Biochemistry 30, 2655-2664), we have demonstrated that the 50-kDa fragment retains a Mg(2+)-dependent DNA polymerizing activity, whereas the 40-kDa polypeptide is able to catalyze the excision of mispaired nucleotides at the 3'-OH terminus of a primer/template DNA substrate in the presence of Mn2+ ions. On the other hand, the 100-kDa protein possess both activities. To date, this is the first report indicating, on the basis of direct functional data, that the polymerization and the 3'-5' exonuclease activity of a family B DNA polymerase can be ascribed to physically distinct modules of the enzyme molecule.
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PMID:Evidence that an archaeal alpha-like DNA polymerase has a modular organization of its associated catalytic activities. 813 6

In this report, we describe the isolation, molecular genetic mapping, and phenotypic characterization of vaccinia virus mutants resistant to cytosine arabinoside (araC) and phosphonoacetic acid (PAA). At 37 degrees C, 8 microM araC was found to prevent macroscopic plaque formation by wild-type virus and to cause a 10(4)-fold reduction in viral yield. Mutants resistant to 8 microM araC were selected by serial passage of a chemically mutagenized viral stock in the presence of drug. Because recovery of mutants required that initial passages be performed under less stringent selective conditions, and because plaque-purified isolates were found to be cross-resistant to 200 micrograms of PAA per ml, it seemed likely that resistance to araC required more than one genetic lesion. This hypothesis was confirmed by genetic and physical mapping of the responsible mutations. PAAr was accorded by the acquisition of one of three G-A transitions in the DNA polymerase gene which individually alter cysteine 356 to tyrosine, glycine 372 to aspartic acid, or glycine 380 to serine. AraCr was found to require one of these substitutions plus an additional T-C transition within codon 171 of the DNA polymerase gene, a change which replaces the wild-type phenylalanine with serine. Congenic viral stocks carrying one of the three PAAr lesions, either alone or in conjunction with the upstream araCr lesion, in an otherwise wild-type background were generated. The PAAr mutations conferred nearly complete resistance to PAA, a slight degree of resistance to araC, hypersensitivity to aphidicolin, and decreased spontaneous mutation frequency. Addition of the mutation at codon 171 significantly augmented araC resistance and aphidicolin hypersensitivity but caused no further change in mutation frequency. Several lines of evidence suggest that the PAAr mutations primarily affect the deoxynucleoside triphosphate-binding site, whereas the codon 171 mutation, lying within a conserved motif associated with 3'-5' exonuclease function, is postulated to affect the proofreading exonuclease of the DNA polymerase.
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PMID:Genetic characterization of the vaccinia virus DNA polymerase: cytosine arabinoside resistance requires a variable lesion conferring phosphonoacetate resistance in conjunction with an invariant mutation localized to the 3'-5' exonuclease domain. 838 30

We have purified a high molecular weight complex (RC-1) from calf thymus nuclei that catalyzes a recombinational repair of double-strand gaps and deletions in DNA by gene conversion as well as cross-over events leading to cointegrant products. These have been detected by polymerase chain reaction analysis using oligonucleotide primer pairs that detect joined sequences originally present on only one or the other of the recombination substrates. RC-1 has an apparent molecular mass of about 550-600 kDa and contains at least five polypeptide chains: molecular masses about 230, 210, 160, 130, and 40 kDa. RC-1 contains a DNA polymerase, identified as DNA polymerase epsilon, that co-purifies with RC-1. A DNA ligase, most likely mammalian DNA ligase III, and a 5'-3' exonuclease also copurify with the RC-1. Most preparations of RC-1 contain low levels of a double-strand endonuclease, 3'-5' exonuclease and single-strand nuclease activities. However, DNA helicase, terminal deoxynucleotidyl transferase, or DNA topoisomerase I and II were not detected in RC-1. The DNA polymerase and DNA ligase in RC-1 can act in concert to repair a multiply gapped DNA to a covalently repaired duplex. The bovine single-strand-binding protein stimulates the formation of the recombination products and the repair reaction mentioned above about 4-fold.
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PMID:A mammalian protein complex that repairs double-strand breaks and deletions by recombination. 839 64

Nickel is a genotoxic carcinogen. However, the mechanisms of nickel-induced genotoxicity are not well understood. We have investigated the effects of Ni2+ ions on DNA polymerase activity and the fidelity of DNA replication in vitro. The effect of Ni2+ on different DNA polymerases is quite variable. The amount of enzyme inhibition and degree of alteration in replication fidelity induced by Ni2+ are dependent both on the polymerase and its associated 3'-5' exonuclease activity. Some polymerases, such as E. coli DNA polymerase I, AMV reverse transcriptase and human DNA polymerase alpha, can utilize Ni2+ as a weak substitute for Mg2+ during DNA replication. Other polymerases are very sensitive to inhibition by Ni2+ and the IC50 can vary by an order of magnitude. T4 polymerase is relatively insensitive to inhibition by Ni2+, although the sensitivity is enhanced in the absence of added Mg2+, and Ni preferentially inhibits the 3'-5' exonuclease function of T7 DNA polymerase. The fidelity and processivity of DNA polymerases may be either increased or decreased by Ni ions in a polymerase dependent manner. The inhibition DNA polymerase activity and altered replication fidelity may contribute significantly to Ni-induced mutagenesis and genotoxicity in vivo.
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PMID:Effects of nickel ions on polymerase activity and fidelity during DNA replication in vitro. 840 77

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.
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PMID:Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli. 841 92


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