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)

DNA mismatch correction is a strand-specific process involving recognition of noncomplementary Watson-Crick nucleotide pairs and participation of widely separated DNA sites. The Escherichia coli methyl-directed reaction has been reconstituted in a purified system consisting of MutH, MutL, and MutS proteins, DNA helicase II, single-strand DNA binding protein, DNA polymerase III holoenzyme, exonuclease I, DNA ligase, along with ATP (adenosine triphosphate), and the four deoxynucleoside triphosphates. This set of proteins can process seven of the eight base-base mismatches in a strand-specific reaction that is directed by the state of methylation of a single d(GATC) sequence located 1 kilobase from the mispair.
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PMID:DNA mismatch correction in a defined system. 266 76

Mutations in the mutY gene of Escherichia coli confer hypermutability reflecting G.C to T.A transversion mutations and result in a deficiency in methyl-independent G-A to G.C mismatch correction. In the present work, the mutY product has been purified to near homogeneity by virtue of its ability to restore G-A to G.C mismatch correction to cell-free extracts of a mutS mutY strain. The 36-kDa protein renders the strand containing the mispaired adenine labile to base-catalyzed cleavage and sensitive to cleavage by several apurinic/apyrimidinic-site endonucleases, with the sites of strand scission by both agents corresponding to the location of the mismatch. These findings indicate that MutY is a DNA glycosylase that hydrolyzes the glycosyl bond linking the mis-paired adenine to deoxyribose. MutY, a 5'-apurinic/apyrimidinic-site endonuclease, DNA polymerase I, and DNA ligase are sufficient to reconstitute MutY-dependent G-A to G.C repair in vitro.
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PMID:Escherichia coli mutY gene encodes an adenine glycosylase active on G-A mispairs. 268 64

The main biochemical determinants involved in cytosine arabinoside (Ara-C) metabolism were studied in one lymphoblastic (Reh) and two myeloid (HL60 and K562) human leukemic cell lines exhibiting various sensitivities to Ara-C, Reh being the most and HL60 the least sensitive. The level of intracellular Ara-C accumulation and Ara-CTP formation was far more important in Reh cells than in myeloid cell lines but was not closely related to deoxycytidine kinase activity or to deoxycytidine triphosphate pool size. The level of Ara-C incorporated into DNA was similar in the three cell lines. Ara-CTP formation correlated better with the cytotoxicity to clonogenic cells than did Ara-C incorporation into DNA. DNA polymerase alpha was moderately inhibited to various degrees, depending on the cell line; this moderate inhibition does not seem sufficient to explain the inhibition of DNA synthesis. The activity of DNA ligase, the enzyme joining the Okazaki fragments, which was not detected in Reh cells, was strongly inhibited by Ara-C in HL60 and to a lesser degree, in K562 cells. The inhibition of DNA ligase probably also contributes to the inhibition of DNA synthesis and, thus, to the cytotoxic effect of Ara-C and may explain the smaller size of DNA fragments observed following Ara-C treatment. The variations in each critical determinant observed in these three cell lines increase the complexity and plurality of the mechanisms of Ara-C action.
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PMID:A study of the mechanisms of cytotoxicity of Ara-C on three human leukemic cell lines. 275 6

1. The structural gene for cholinephosphate cytidylyltransferase (CCT) was isolated from a Saccharomyces cerevisiae genomic library by means of complementation in a mutant of the yeast defective in the enzyme. The cloned DNA restored both the growth and cholinephosphate cytidylyltransferase activity of the mutant. Whereas the enzyme of the mutant was thermolabile, the enzyme produced by the transformant was indistinguishable in heat stability from that produced by the wild type. 2. Strains carrying a multicopy recombinant plasmid overproduced cholinephosphate cytidylyltransferase. The overproduction of the enzyme brought about an increase in the synthesis of CDPcholine in the transformant, but there was no increase in the overall rate of phosphatidylcholine synthesis. 3. The cloned DNA was subcloned into a 2.5-kb DNA fragment. The nucleotide sequence which contained CCT was determined by the dideoxy chain-termination method. The sequence contained an open reading frame capable of encoding a protein of 424 amino acid residues with a calculated relative molecular mass of 49,379.31. Northern blot analysis showed that this DNA segment is transcribed in yeast cells and the length of the transcript is consistent with the putative translation product. 4. Hydropathy analysis according to Kyte and Doolittle indicated that the primary translation product contains extended hydrophilic stretches in its N- and C-terminal regions. 5. The primary translation product contains a region showing local sequence homology with nucleotidyl-transfer enzymes such as DNA polymerase (Escherichia coli), CDPdiacylglycerol pyrophosphatase (E. coli), 3-deoxy-manno-octulosonate cytidylyltransferase (E. coli) and DNA ligase (T4 phage), suggesting that these five enzymes are evolutionarily related. Statistically significant sequence homology was also noted between the human c-fos gene product and the enzyme.
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PMID:Molecular cloning and characterization of the gene encoding cholinephosphate cytidylyltransferase in Saccharomyces cerevisiae. 282 47

A simple method for generating cDNA libraries has been described (1) in which RNase H-DNA polymerase I-mediated second-strand cDNA synthesis primes from an RNA oligonucleotide derived from the 5' (capped) end of mRNA. The size of this oligonucleotide and the fate of the information corresponding to the RNA during subsequent cloning have not been established. We show here that the 5'-most RNA primer varies in length from 8 to 21 nucleotides, and that information corresponding to the length of the RNA primer is normally lost during cloning. A modification of the second-strand cDNA synthesis procedure is described which allows cloning of all, or almost all, of the primer sequence information. In addition, we show that the presence of E. coli DNA ligase during second-strand cDNA synthesis can increase the length of the cDNA clones obtained from long RNAs. Cloning by addition of linkers provides the greatest chance of obtaining near full-length cDNA clones from long mRNAs.
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PMID:Second-strand cDNA synthesis with E. coli DNA polymerase I and RNase H: the fate of information at the mRNA 5' terminus and the effect of E. coli DNA ligase. 283 25

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.
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PMID:Complete enzymatic synthesis of DNA containing the SV40 origin of replication. 284 39

We have studied homologous recombination in a derivative of phage lambda containing two 1.4-kb repeats in inverted orientation. Inversion of the intervening 2.5-kb segment occurred efficiently by the Escherichia coli RecBC pathway but markedly less efficiently by the lambda Red pathway or the E. coli RecE or RecF pathways. Inversion by the RecBCD pathway was stimulated by Chi sites located to the right of the invertible segment; this stimulation decreased exponentially by a factor of about 2 for each 2.2 kb between the invertible segment and the Chi site. In addition to RecA protein and RecBCD enzyme, inversion by the RecBC pathway required single-stranded DNA binding protein, DNA gyrase, DNA polymerase I and DNA ligase. Inversion appeared to occur either intra- or intermolecularly. These results are discussed in the framework of a current molecular model for the RecBC pathway of homologous recombination.
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PMID:Genetic functions promoting homologous recombination in Escherichia coli: a study of inversions in phage lambda. 295 Dec 95

An easy and quick method to synthesize large cDNA molecules and to clone them with very high efficiency in the expression vector lambda gt11 is described. The technique employs RNase H and Escherichia coli DNA ligase treatment during second-strand synthesis, followed by repair of the ds cDNA extremities by S1 nuclease and PolIk (Klenow fragment) treatment. This treatment allows efficient addition of suitable linkers and results in a 100-fold increase in the yield of cloned cDNA, when compared with other published techniques. Using 75 ng of poly(A)+ RNA from CHO cells, we have prepared a library of 1.1 X 10(7) clones. This library was screened with polyclonal antibodies raised against a 100-kDal nucleolar protein of CHO cells. Five recombinants were isolated with inserts of 500-2500 bp. The average size of cDNA obtained by this method is considerable: the 2500-bp cDNA represents 90% of the mRNA coding for the 100-kDal protein.
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PMID:A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein. 299 85

Escherichia coli strains containing mutations in various deoxyribonucleic acid synthesis cistrons have been tested for their ability to support bacteriophage N4 growth and, specifically, N4 DNA synthesis. N4 DNA synthesis is independent of the activity of the products of the E. coli dnaA, dnaB, dnaC, dnaE, dnaG, and rep genes. In contrast, N4 DNA replication requires the products of the dnaF, (ribonucleotide reductase) and lig (DNA ligase) genes of E. coli. N4 DNA replication, specifically processing of short DNA fragments requires the 5'-3' exonuclease activity of the polA gene product. However, its DNA polymerizing activity is not required. In addition, the sensitivity of N4 DNA synthesis to inhibitors or temperature-sensitive mutants of E. coli DNA gyrase suggests that this activity is required for N4 DNA synthesis. To date, we have found five N4 gene products required for N4 DNA replication: dbp (a single-stranded DNA binding protein), dnp (a DNA polymerase), dns (unknown function), vRNAp (the N4 virion-associated, DNA-dependent RNA polymerase) and exo (a 5'-3' exonuclease).
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PMID:Host and phage-coded functions required for coliphage N4 DNA replication. 300 44

A number of enzymes thought to be involved in DNA replication have been identified in the brain. These include single-stranded DNA-binding proteins, topoisomerases I and II, DNA polymerase alpha, a protein that binds Ap4A and might be classified as a DNA polymerase alpha accessory protein, RNase H, DNA polymerase beta, DNA ligase, an endo- and an exonuclease of unknown function, DNA methyl transferase and poly(ADPR) synthase. In contrast, little is known about the enzymology of DNA repair in brain. The few enzymes identified comprise uracil-DNA glycosylase, DNA polymerase beta, DNA polymerase alpha (which in neurons is present only at immature stages), DNA ligase, poly(ADPR) synthase, and O6-alkylguanine-DNA alkyltransferase. In addition, an exonuclease acting on depurinated single-stranded DNA (tentatively listed here as 3'----5' exonuclease), an endonuclease of unknown function as well as ill-defined acid and alkaline deoxyribonucleases also occur in brain.
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PMID:Enzymology of DNA replication and repair in the brain. 300 64


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