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)

The thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) has been truncated to molecular regions essential for polymerase activity. Two truncated forms of the full-length 832 amino acid Taq polymerase have been constructed according to sequence alignments and the known domain structure of the homologous Escherichia coli DNA polymerase I (E.coli pol I): variant delta288 (lacking the N-terminal 288 amino acid portion) and variant delta413 (lacking the N-terminal 413 amino acid portion). Both protein fragments were stable and showed polymerase activity, albeit specific activity and thermostability of the variant delta413 were significantly decreased compared with the full length Taq polymerase. In order to increase the thermostability of the variant delta413, a three-dimensional model of the polymerase domain of Taq polymerase was built by homology with a model of the Klenow fragment of the E.coli pol I based on the available Calpha coordinates. Consequently two variants were designed and constructed using site-directed mutagenesis. The strategies used were deletion of 10 flexible amino acids and replacement of two hydrophobic amino acids on the surface by more hydrophilic ones. Compared with the initial protein fragment, both variant enzymes showed an increase in polymerase activity and thermostability. After the completion of this work, X-ray coordinates of the Taq polymerase became available from the protein structure data bank. A comparison between the homology model and the experimental three-dimensional structure proved the quality of the model.
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PMID:Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments. 951 16

The bacteria Escherichia coli contains several exonucleases acting on both double- and single-stranded DNA and in both a 5'-->3' and 3'-->5' direction. These enzymes are involved in replicative, repair and recombination functions. We have identified a new exonuclease found in E.coli, termed exonuclease IX, that acts preferentially on single-stranded DNA as a 3'-->5' exonuclease and also functions as a 3'-phosphodiesterase on DNA containing 3'-incised apurinic/apyrimidinic (AP) sites to remove the product trans -4-hydroxy-2-pentenal 5-phosphate. The enzyme showed essentially no activity as a deoxyribophosphodiesterase acting on 5'-incised AP sites. The activity was isolated as a glutathione S-transferase fusion protein from a sequence of the E.coli genome that was 60% identical to a 260 bp region of the small fragment of the DNA polymerase I gene. The protein has a molecular weight of 28 kDa and is free of AP endonuclease and phosphatase activities. Exonuclease IX is expressed in E.coli , as demonstrated by reverse transcription-PCR, and it may function in the DNA base excision repair and other pathways.
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PMID:Exonuclease IX of Escherichia coli. 959 42

The RAD27/RTH1 gene of Saccharomyces cerevisiae encodes a structural and functional homolog of the 5'-3' exonuclease function of Escherichia coli DNA polymerase I. Four alleles of RAD27 were recovered in a screen for hyper-recombination, a phenotype also displayed by polA mutants of E.coli. All four rad27 mutants showed similar high levels of mitotic recombination, but varied in their growth rate at various temperatures, and sensitivity to the DNA damaging agent methyl methane sulfonate. Dependence of viability of rad27 strains on recombination was determined by crossing a strain containing a null allele of RAD27 to strains containing a mutation in either the RAD1, RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11, XRS2 or RAD59 gene. In no case were viable spore products recovered that contained both mutations. Elimination of the non-homologous end-joining pathway did not affect the viability of a rad27 strain. This suggests that lesions generated in the absence of RAD27 must be processed by homologous recombination.
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PMID:Homologous recombination is required for the viability of rad27 mutants. 983 87

Four kinds of oxidatively damaged DNA precursors, 8-hydroxydeoxyguanosine 5'-triphosphate (8-OH-dGTP), 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), 5-hydroxydeoxycytidine 5'-triphosphate (5-OH-dCTP) and 5-formyldeoxyuridine 5'-triphosphate (5-CHO-dUTP), were employed in in vitro gap-filling reactions of the supF gene conducted by the Escherichia coli DNA polymerase III holoenzyme, and these treated DNAs were transfected into various E.coli strains. When the manipulated DNAs were transfected into the repair-proficient strain, supF mutants were obtained much more frequently by the purine nucleotides than by the pyrimidine nucleotides (2-OH-dATP > 8-OH-dGTP >> 5-OH-dCTP approximately 5-CHO-dUTP). This result is in contrast to our previous observation that these four oxidatively damaged nucleotides induce chromosomal gene mutations with similar frequencies when incorporated directly into E.coli. 2-OH-dATP elicited G-->T transversions, indicating the formation of G*2-OH-dATP pairs. These results demonstrate that 2-OH-dATP was highly mutagenic in this assay system containing the in vitro DNA synthesis by the E.coli replicative DNA polymerase, in addition to in the in vivo assay system reported previously. Slight increases in the mutant frequencies were observed when alkA (for 8-OH-dGTP and 2-OH-dATP) and mutY (for 2-OH-dATP) strains were used as hosts. This is the first report that clearly shows the formation of G*2-OH-dATP pairs.
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PMID:2-Hydroxy-dATP is incorporated opposite G by Escherichia coli DNA polymerase III resulting in high mutagenicity. 1071 Apr 31

Cytosine residues of poly(C) and heat-denatured calf thymus DNA were transformed into 5,6-dihydrouracil-6-sulfonate (U(SO(-) (3))) residues by treatment with bisulfite. The poly(U(SO(-) (3))(2), C(3)) and poly(U(SO(-) (3))(9), C(1)) prepared did not form inter-base binding with either poly(A) or poly(I) as judged by the absence of hypochromicity in ultraviolet absorbance. U(SO(-) (3)) residues in the DNA inactivated it to serve as template for E.coli DNA polymerase I, while the template activity was restored by conversion of the U(SO(-) (3)) residues into U.
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PMID:The effect of bisulfite modification on the template activity of DNA for DNA polymerase I. 1079 21

Among the polymerases, DNA polymerase alpha-primase is involved in lagging strand DNA synthesis. A previous report indicated that DNA polymerase alpha-primase initiates primer RNA synthesis with purine bases on a single-stranded G-rich telomere repeat. In this study, we found that DNA polymerase alpha-primase precisely initiated with adenosine opposite the 3'-side thymidine in the G-rich telomere repeat 5'-(TTAGGG)(n)-3' under rATP-rich conditions. Then, DNA polymerase alpha-primase synthesized the nascent DNA fragments by extending the primer. It was remarkable that DNA polymerase alpha-primase further expanded the product DNA far beyond the length of the template DNA, as ladders of multiple hexanucleotides on polyacrylamide gel electrophoresis. Using an oligomer duplex 5'-A(GGGTTA)(5)-3'/5'-(TAACCC)(5)T-3' as a template-primer, we show that both the Klenow fragment of Escherichia coli DNA polymerase I and HIV reverse transcriptase could expand telomere DNA sequences as well, giving products greater than the size of the template DNA. The maximum product lengths with these polymerases were approximately 40-90 nt longer than the template length. Our data imply that DNA polymerases have an intrinsic activity to expand the hexanucleotide repeats of the telomere sequence by a slippage mechanism and that DNA polymerase alpha uses both the repeat DNA primers and the de novo RNA primers for expansion. On the other hand, a plasmid harboring a eukaryotic telomere repeat showed remarkable genetic instability in E.coli. The telomere repeats exhibited either expansions or deletions by multiple hexanucleotide repeats during culture for a number of generations, suggesting involvement of the slippage mechanism in the instability of telomeric DNA in vivo.
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PMID:In vitro expansion of mammalian telomere repeats by DNA polymerase alpha-primase. 1093 27

The intervening domain of the thermostable Thermus aquaticus DNA polymerase (TAQ: polymerase), which has no catalytic activity, has been exchanged for the 3'-5' exonuclease domain of the homologous mesophile Escherichia coli DNA polymerase I (E.coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (TNE: polymerase). Three chimeric DNA polymerases have been constructed using the three-dimensional (3D) structure of the Klenow fragment of the E.coli pol I and 3D models of the intervening and polymerase domains of the TAQ: polymerase and the TNE: polymerase: chimera TaqEc1 (exchange of residues 292-423 from TAQ: polymerase for residues 327-519 of E.coli pol I), chimera TaqTne1 (exchange of residues 292-423 of TAQ: polymerase for residues 295-485 of TNE: polymerase) and chimera TaqTne2 (exchange of residues 292-448 of TAQ: polymerase for residues 295-510 of TNE: polymerase). The chimera TaqEc1 showed characteristics from both parental polymerases at an intermediate temperature of 50 degrees C: high polymerase activity, processivity, 3'-5' exonuclease activity and proof-reading function. In comparison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'-5' exonuclease activity and no proof-reading function. The chimera TaqTne1 showed an optimum temperature at 60 degrees C, decreased polymerase activity compared with the TAQ: polymerase and reduced processivity. The chimera TaqTne2 showed high polymerase activity at 72 degrees C, processivity and less reduced thermostability compared with the chimera TaqTne1.
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PMID:Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase. 1105 59

Endonuclease III from Escherichia coli, yeast (yNtg1p and yNtg2p) and human and E.coli endonuclease VIII have a wide substrate specificity, and recognize oxidation products of both thymine and cytosine. DNA containing single dihydrouracil (DHU) and tandem DHU lesions were used as substrates for these repair enzymes. It was found that yNtg1p prefers DHU/G and exhibits much weaker enzymatic activity towards DNA containing a DHU/A pair. However, yNtg2p, E. coli and human endonuclease III and E.coli endonuclease VIII activities were much less sensitive to the base opposite the lesion. Although these enzymes efficiently recognize single DHU lesions, they have limited capacity for completely removing this damaged base when DHU is present on duplex DNA as a tandem pair. Both E.coli endonuclease III and yeast yNtg1p are able to remove only one DHU in DNA containing tandem lesions, leaving behind a single DHU at either the 3'- or 5'-terminus of the cleaved fragment. On the other hand, yeast yNtg2p can remove DHU remaining on the 5'-terminus of the 3' cleaved fragment, but is unable to remove DHU remaining on the 3'-terminus of the cleaved 5' fragment. In contrast, both human endonuclease III and E.coli endonuclease VIII can remove DHU remaining on the 3'-terminus of a cleaved 5' fragment, but are unable to remove DHU remaining on the 5'-terminus of a cleaved 3' fragment. Tandem lesions are known to be generated by ionizing radiation and agents that generate reactive oxygen species. The fact that these repair glycosylases have only a limited ability to remove the DHU remaining at the terminus suggests that participation of other repair enzymes is required for the complete removal of tandem lesions before repair synthesis can be efficiently performed by DNA polymerase.
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PMID:Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII. 1113 10

The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets.
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PMID:Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure. 1137 66

A site-specifically modified oligonucleotide containing a single 2'-deoxyribonolactone lesion was used as a template for primer extension reactions catalyzed by M-MuLV reverse transcriptase (RT) and by the Klenow fragments of Escherichia coli DNA polymerase proficient (KF exo(+)) or deficient (KF exo(-)) in exonuclease activity. Analysis of the extension products in the presence of the four dNTPs or of a single dNTP showed that the M-MuLV RT was completely blocked and did not incorporate any dNMP opposite 2'-deoxyribonolactone. KF exo(-) preferentially incorporated nucleotides opposite the lesion following the frequency order dAMP > dGMP >> dTMP approximately dCMP and thus appeared to obey the 'A rule' for preferential incorporation as has been shown previously for the 2'-deoxyribose abasic site. In the sequence context examined, the primer extension by KF exo(-) appeared to be less efficient when dAMP was positioned opposite the lesion as compared with dTMP or dGMP. These two nucleotides promoted a more efficient polymerization accompanied by nucleotide deletion through misalignment incorporations. We therefore predict that the sequence context may strongly influence the translesional synthesis by KF exo(-) and thus the miscoding and mutational potential of the 2'-deoxyribonolactone in E.coli.
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PMID:Translesional synthesis on DNA templates containing the 2'-deoxyribonolactone lesion. 1143 17


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