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:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Excision repair of UV-damaged Bacillus subtilis transforming DNA has been carried out by a sequential enzyme system in vitro. Incision adjacent to the pyrimidine dimer in the DNA strand by correndonuclease II-initiated excision of the damage by the 5' in equilibrium 3'-directed exonuclease of the Micrococcus luteus DNA polymerase. Reinsertion of nucleotides into the gap in the strand by the DNA polymerase at 10 degrees C terminated in a single-strand break which was sealed by a polynucleotide ligase, thereby repairing the DNA strand. This restored biological activity to damaged DNA up to doses resulting in 60% inactivation of transforming activity. At higher doses, less repair was achieved, due to the development of double-strand breaks during the in vitro incision and excision steps.
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PMID:Enzymatic repair of UV-irradiated DNA in vitro. 81 Dec 6

We have studied excision-repair of UV-irradiated phiX174 RFI DNA in vitro with UV-specific endonuclease from Micrococcus luteus (UV-endo), DNA polymerase I from Escherichia coli and DNA ligase from phage T4 infected E. coli. Excision-repair was measured a) by physico-chemical methods, i.e. by determination of the conversion of RF I DNA into RF II DNA by UV-endo and by the subsequent conversion of RF II DNA ligase, b) by biological methods i. e. by measuring the ability of the reaction product to form phages upon incubation with spheroplasts from the appropriate strains of E. coli. Using the first method, we have shown, that more than 90% of the pyrimidine dimers can be repaired in vitro; with the latter method we have shown, that the molecules which are repaired as defined by method a) have regained full biological activity. Exonuclease III was found to be not essential for excision-repair in vitro and also did not stimulate repair. From this result we conclude that UV-endo generates 3'OH endgroups, in agreement with results obtained by Hamilton et al. (1974). The usefulness of the method presented in this paper with regard to the study of excision-repair is discussed.
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PMID:Physico-chemical and biological study of excision-repair of UV--irradiated phiX174 RF DNA in vitro. 105 35

An endonuclease from Escherichia coli which acts specificially upon UV-irradiated DNA (correndonuclease II) and is absent from the uvrA and uvrB mutants has been isolated and partially chacterized. The enzyme is present in normal amounts in the urvC mutant. It elutes from phosphocellulose at about 0.25 M potassium phosphate (pH 7.5) and passes through dialysis tubing. The enzyme binds tightly to UV-irradiated DNA but does not bind to unirradiated DNA. The enzyme incises irradiated DNA to the 5' side of a pyrimidine dimer and leaves a 5'-phosphoryl terminus which can be resealed with polynucleotide ligase. The Km of the enzyme is about 1.5 X 10(-8) M dimers. Endonucleolytic activity of the enzyme is inhibited by caffeine with a KI of about 10mM.
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PMID:The Escherichia coli UV endonuclease (correndonuclease II). 110 24

The size of the repair patch produced by E. coli DNA polymerase (Pol I) following the removal of a pyrimidine dimer from DNA in response to the nicking activity of T4 endonuclease (T4 endo V) was determined. A 48-bp DNA containing a pyrimidine dimer at a defined location was labelled in the damaged strand and incubated with T4 endo V and E. coli endonuclease IV. Subsequently, DNA synthesis by DNA Pol I was carried out in the presence of four dNTPs, ATP and DNA ligase. Analysis of the reaction products on a sequencing gel revealed a ladder of only 4-oligonucleotides, 1-4 nucleotides greater in length than the fragment generated by the combined nicking activities of T4 endo V and E. coli endonuclease IV. Thus we conclude that the in vitro repair patch size of T4 endo V is 4 nucleotides and that in some cases the repaired DNA is not ligated.
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PMID:In vitro characterization of repair synthesis initiated by T4 endonuclease V on a synthetic DNA substrate. 151 8

The phage T4 denV gene, coding for the pyrimidine-dimer specific T4 endonuclease V, was transfected into human repair-proficient fibroblasts, repair-deficient xeroderma pigmentosum fibroblasts, and into wild type CHO hamster cells. Transfectants maintained denV DNA and expressed denV mRNA. Purified T4 endonuclease V encapsulated in liposomes was also used to treat repair-proficient and -deficient human cells. The denV transfected clones and liposome-treated cells showed increased unscheduled DNA synthesis and enhanced removal of pyrimidine dimers compared to controls. Both denV gene transfection and endonuclease V liposome treatment enhanced post-UV survival in xeroderma pigmentosum cells but had no effect on survival in repair-proficient human or hamster cells. The results demonstrate that an exogenous DNA repair enzyme can correct the DNA repair defect in xeroderma pigmentosum cells and enhance DNA repair in normal cells.
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PMID:Enhancement of ultraviolet-DNA repair in denV gene transfectants and T4 endonuclease V-liposome recipients. 166 12

The phr gene of Escherichia coli K-12 encodes the light-dependent, DNA repair enzyme photolyase, which removes UV light-induced pyrimidine dimers from cellular DNA. From Southern hybridization analysis of several strains containing successively extended phr deletions, we have determined the direction of transcription of the phr gene on the E. coli K-12 chromosome. Northern (RNA) hybridization analysis suggests that the phr gene is cotranscribed with a previously identified gene of unknown function (orf169) into two messages of different lengths. S1 nuclease mapping analysis indicates that the two transcripts share a single termination site but initiate at two different sites. Finally, we have determined that the presence of orf169 is not necessary for phr gene activity in vivo.
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PMID:Physical analysis of phr gene transcription in Escherichia coli K-12. 169 32

Endonuclease V, a pyrimidine dimer-specific DNA repair enzyme, was chemically modified by reductive methylation, a technique that specifically methylates primary amino groups. Upon reaction of endonuclease V with [14C]formaldehyde (14CH2O) in the presence of the reducing agent sodium cyanoborohydride (Na-CNBH3), it was discovered that 0.8 methylation/endonuclease V molecule was required to reduce both the glycosylase and the phosphodiester lyase activities by 70-80%. Pyrimidine dimer-specific binding was not eradicated at a level of methylation equivalent to 0.8 CH3/endonuclease V molecule but was eradicated at higher levels of methylation. Endonuclease V that had been modified with an average of 1.6 CH3/molecule was digested with Staphylococcus aureus strain V8 protease and the peptides subsequently separated by reverse-phase high performance liquid chromatography. Radiolabel was found exclusively on the peptide including the amino terminus, as determined by the percent amino acid composition. Neither intact CH3-endonuclease V nor radiolabeled peptides were able to be sequenced by Edman degradation indicating blockage of the amino terminus by methylation. This study shows strong evidence for the unusual involvement of the alpha NH2 moiety in the chemical mechanisms of endonuclease V. A reaction mechanism that incorporates these findings is presented.
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PMID:Reductive methylation of the amino terminus of endonuclease V eradicates catalytic activities. Evidence for an essential role of the amino terminus in the chemical mechanisms of catalysis. 189 43

T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function by virtue of its ability to function in the presence of metal-chelating agents. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer.
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PMID:Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding. 203 8

Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.
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PMID:Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair. 240 55


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