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)

Mutants of Escherichia coli having reduced levels of exonuclease VII activity have been isolated by a mass screening procedure. Nine mutants, five of which are known to be of independent origin, were obtained and designated xse. The defects in these strains lie at two or more loci. One of these loci, xseA, lies in the interval between purG and purC; it is 93 to 97% co-transducible with guaA. The order of the genes in this region is purG-xseA guaA,B-purC. The available data do not allow xseA to be ordered with respect to guaA,B. Exonuclease VII purified from E. coli KLC3 xseA3 is more heat labile than exonuclease VII purified from the parent, E. coli PA610 xse+. Therefore, xseA is the structural gene for exonuclease VII. Mutants with defects in the xseA gene show increased sensitivity to nalidixic acid and have an abnormally high frequency of recombination (hyper-Rec phenotype) as measured by the procedure of Konrad and Lehlman (1974). The hyper-Rec character of xseA strains is approximately one-half that of the polAex1 mutant defective in the 5' leads to 3' hydrolytic activity of deoxyribonucleic acid polymerase I. The double mutant, polAex1 xseA7, is twice as hyper-Rec as the polAex1 mutant alone. The xseA- strains are slightly more sensitive to ultraviolet irradiation than the parent strain. Bacteriophages T7, fd, and lambdared grow normally in xseA- strains.
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PMID:Escherichia coli mutants deficient in exonuclease VII. 32 Jan 98

Py pyrimidine dimers Py correndonucleases I and II from Micrococcus luteus act exclusively on thymine-thymine, cytosine-cytosine, and thymine-cytosine cyclobutyl dimers in DNA, catalyzing incision 5' to the damage and generating 3'-hydroxyl and 5'-phosphoryl termini. Both enzymes initiate excision of pyrimidine dimers in vitro by correxonucleases and DNA polymerase I. The respective incised DNAs, however, differ in their ability to act as substrate for phage T4 polynucleotide ligase or bacterial alkaline phosphatase, suggesting that each endonuclease is specific for a conformationally unique site. The possibility that their respective action generates termini which represent different degrees of single strandedness is suggested by the unequal protection by Escherichia coli binding protein from the hydrolytic action of exonuclease VII.
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PMID:Micrococcus luteus correndonucleases. II. Mechanism of action of two endonucleases specific for DNA containing pyrimidine dimers. 33 May 26

An isogenic series of Escherichia coli strains deficient in various combinations of three 5' leads to 3' exonucleases (exonuclease V, exonuclease VII, and the 5' leads to 3' exonuclease of DNA polymerase I) was constructed and examined for the ability to excise pyrimidine dimers after UV irradiation. Although the recB and recC mutations (deficient in exonuclease V) proved to be incompatible with the polA(Ex) mutation (deficient in the 5' leads to 3' exonuclease of DNA polymerase I), it was possible to reduce the level of the recB,C exonuclease by the use of temperature-sensitive recB270 recC271 mutants. It was found that, by employing strains deficient in exonuclease V, postirradiation DNA degradation could be reduced and dimer excision measurements could be facilitated. Mutants deficient in exonuclease V were found to excise dimers at a rate comparable to that of the wild type. Mutants deficient in exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I are slightly slower than the wild type at removing dimers accumulated after doses in excess of 40 J/m2. However, although strains with reduced levels of exonuclease VII excised dimers at the same rate as the wild type, the addition of an exonuclease VII deficiency to a strain with reduced levels of exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I caused a marked decrease in the rate and extent of dimer excision. These observations support previous indications that the 5' leads to 3' exonuclease of DNA polymerase I is important in dimer removal and also suggest a role for exonuclease VII in the excision repair process.
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PMID:Pyrimidine dimer excision in Escherichia coli strains deficient in exonucleases V and VII and in the 5' leads to 3' exonuclease of DNA polymerase I. 36 15

Procedures have been worked out for Aspergillus nuclease S1 and mung been nuclease to quantitatively cleave off both of the 12-nucleotide long, single-stranded cohesive ends of lambdaDNA. This cleavage is indicated by the almost complete elimination of the repair incorporation of radioactive nucleotides by DNA polymerase into the digested DNA. With S1 nuclease, cleavage was complete at 10 degrees as well as at 30 degrees. Under the conditions for quantitative cleavage of the single-stranded regions there was no digestion of the double-stranded lambdaDNA. The mung bean nuclease cleaved off the cohesive ends completely at 30 degrees but at 5 degrees, the cleavage was not complete even at high enzyme concentration. The nearest neighbor analysis of the repaired DNA indicates that at 5 degrees about four nucleotides remained undigested. The mung bean nuclease also introduced, under the conditions used, some nicks into double-stranded DNA as determined by the repair incorporation. The Escherichia coli exonuclease VII cleaved off part of the cohesive ends of lambdaDNA, leaving two nucleotides on each end as single-stranded tails.
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PMID:Specific hydrolysis of the cohesive ends of bacteriophage lambda DNA by three single strand-specific nucleases. 114 Dec 22

The conservation of the herpesvirus DNA polymerases has allowed cross-hybridization studies to be used for their identification and mapping on the viral genome. With the use of a DNA fragment containing the DNA polymerase gene of human cytomegalovirus (HCMV) as a hybridization probe, we were able to localize the DNA polymerase gene of murine cytomegalovirus (MCMV) to a region within MCMV EcoRI fragment B which spans the HindIII site separating HindIII fragments D and H. This site is colinear with the HCMV strain AD169 DNA polymerase gene. To confirm that this region encoded the MCMV DNA polymerase gene, we sequenced a 5131 nucleotide fragment from the PstI site in HindIII fragment D to a BglII site in HindIII fragment H. Initiating in HindIII fragment D and extending into HindIII fragment H was a long open reading frame (ORF) 1097 amino acids in length with extensive homology to the DNA polymerases of HCMV, herpes simplex virus, and Epstein-Barr virus. Upstream of the polymerase ORF was a reading frame with considerable homology to the carboxy terminal half of the glycoprotein B gene of human herpesviruses. At early times in the infection, we could detect with a probe representing part of the polymerase ORF two 3' coterminal transcripts, 3.9 kb and 1.7 kb in length. S1 nuclease and exonuclease VII analyses indicated that both transcripts were unspliced and initiated at independent sites in HindIII fragment D. By primer extension, we were able to map precisely the 5' end of the 3.9-kb RNA to a site 186 nucleotides upstream of the beginning of the DNA polymerase ORF.
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PMID:Transcription analysis and sequence of the putative murine cytomegalovirus DNA polymerase gene. 171 83

The influence of a C----G transversion at position 1 of the 30-base pair replication origin of bacteriophage phi X174 replicative form I DNA (phi X RFI) was examined in the RF----single-stranded circular DNA replication pathway catalyzed by the combined action of the purified phi X A protein, the Escherichia coli DNA polymerase III holoenzyme, rep helicase, and single-stranded DNA binding protein (Eisenberg, S., Scott, J.F., and Kornberg, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1594-1597; Reinberg, D., Zipursky, S.L., and Hurwitz, J. (1981) J. Biol. Chem. 256, 13143-13151). RFI DNA containing this transversion was cleaved to RFII by the phi X A protein as effectively as DNA containing the wild-type origin. The altered duplex DNA, however, supported replication at a slower rate (3- to 4-fold) than the wild-type DNA due to a defect in the termination and reinitiation reactions catalyzed by the phi X A protein. This defect resulted in the accumulation of DNA products containing long single strands covalently joined to the mutant DNA. These single strands were susceptible to nuclease S1 and exonuclease VII attack. The defect in the template DNA containing C----G transversion was not corrected when this mutant origin was placed on the same strand with a wild-type origin. This double-origin DNA was also replicated poorly and led to the accumulation of large products, in contrast to the products formed with RFI DNA containing two wild-type 30-base pair replication origins on the same strand.
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PMID:Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. I. Replication of DNA containing an alteration in position 1 of the 30-nucleotide icosahedral bacteriophage origin. 297 11

Methyl-directed mismatch repair is initiated by the mismatch-provoked, MutHLS-dependent cleavage of the unmodified strand at a hemimethylated d(GATC) sequence. This reaction is independent of the polarity of the unmodified strand and can occur either 3' or 5' to the mismatch on the unmethylated strand (Au, K. G., Welsh, K., and Modrich, P. (1992) J. Biol. Chem. 267, 12142-12148). The overall repair reaction also occurs without regard to polarity of the unmethylated strand. Both hemimethylated configurations of a linear heteroduplex containing a single d(GATC) sequence are subject to methyl-directed correction in Escherichia coli extracts and in a purified repair system. Repair of both heteroduplex orientations requires MutH, MutL, MutS, DNA helicase II, SSB, and DNA polymerase III holoenzyme, but the two substrates differ with respect to exonuclease requirements for correction. When the unmethylated d(GATC) sequence that directs repair is located 5' to the mismatch on the unmodified strand, mismatch correction requires the 5'--> 3' hydrolytic activity of exonuclease VII or RecJ exonuclease. Repair directed by an unmodified d(GATC) sequence situated 3' to the mismatch depends on the 3'--> 5' activity of exonuclease I. Specific requirements for these activities are evident with circular heteroduplexes containing a single asymmetrically placed d(GATC) sequence, with the requirement for a 5'--> 3' or 3'--> 5' hydrolytic activity being determined by the orientation of the unmethylated strand along the shorter path joining the two sites in the DNA circle. This observation suggests that the methyl-directed repair system utilizes the proximal d(GATC) sequence to direct correction. To our knowledge, these experiments represent the first instance in which exonuclease I, exonuclease VII, and RecJ have been implicated in a particular DNA metabolic pathway.
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PMID:Methyl-directed mismatch repair is bidirectional. 838 65

DNA-DNA interstrand cross-links are thought to be important for the cytotoxicity of many chemotherapeutic agents. To study this more definitively, adduct site-specific methods are used to construct a plasmid with a single nitrogen mustard interstrand cross-link (inter-HN2-pTZSV28). Replication efficiency (RE = [colonies from (inter-HN2-pTZSV28)/(control with no cross-link)]) is approximately 0.3 following transformation into Escherichia coli, implying that the cross-link is repaired. The commonly accepted pathway for cross-link repair, which involves both nucleotide excision repair (NER) and recombination, is ruled out since RE is approximately 0.3 in a delta recA strain. Non-RecA-directed recombination such as copy-choice is also unlikely. However, NER is involved since RE was approximately 0.02 in strains deficient in NER. Base excision repair is not important since RE is approximately 0.3 in strains deficient in 3-methyladenine DNA glycosylases I and II, FAPY DNA glycosylase, both known apurinic/apyrimidinic endonucleases, or DNA deoxyribophosphodiesterase. Another hypothetical repair pathway hinging on a 5' --> 3' exonuclease activity is unlikely since RE is approximately 0.3 in cells deficient in either the 5' --> 3' exonuclease activities of DNA polymerase I, exonuclease VII, or RecJ. Thus, aside from NER, it is unclear what else participates in this recombination-independent repair pathway, although a pathway involing NER followed by replicative bypass of the lesion is the current working hypothesis. Psoralen interstrand cross-links appear not to be repairable by this second pathway, which may have implications for the relative cytotoxicity of interstrand cross-links from different agents.
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PMID:Evidence for a recombination-independent pathway for the repair of DNA interstrand cross-links based on a site-specific study with nitrogen mustard. 913

DNA exonucleases are critical for DNA replication, repair, and recombination. In the bacterium Escherichia coli there are 14 DNA exonucleases including exonucleases I-IX (including the two DNA polymerase I exonucleases), RecJ exonuclease, SbcCD exonuclease, RNase T, and the exonuclease domains of DNA polymerase II and III. Here we report the discovery and characterization of a new E. coli exonuclease, exonuclease X. Exonuclease X is a member of a superfamily of proteins that have homology to the 3'-5' exonuclease proofreading subunit (DnaQ) of E. coli DNA polymerase III. We have engineered and purified a (His)(6)-exonuclease X fusion protein and characterized its activity. Exonuclease X is a potent distributive exonuclease, capable of degrading both single-stranded and duplex DNA with 3'-5' polarity. Its high affinity for single-strand DNA and its rapid catalytic rate are similar to the processive exonucleases RecJ and exonuclease I. Deletion of the exoX gene exacerbated the UV sensitivity of a strain lacking RecJ, exonuclease I, and exonuclease VII. When overexpressed, exonuclease X is capable of substituting for exonuclease I in UV repair. As we have proposed for the other single-strand DNA exonucleases, exonuclease X may facilitate recombinational repair by pre-synaptic and/or post-synaptic DNA degradation.
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PMID:Exonuclease X of Escherichia coli. A novel 3'-5' DNase and Dnaq superfamily member involved in DNA repair. 1051 96