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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

T4 endonuclease V is a pyrimidine dimer-specific endonuclease which generates incisions in DNA at the sites of pyrimidine dimers by a processive reaction mechanism. A model is presented in which the degree of processivity is directly related to the efficacy of the one-dimensional diffusion of endonuclease V on DNA by which the enzyme locates pyrimidine dimers. The modulation of the processive nicking activity of T4 endonuclease V on superhelical covalently closed circular DNA (form I) which contains pyrimidine dimers has been investigated as a function of the ionic strength of the reaction. Agarose gel electrophoresis was used to separate the three topological forms of the DNA which were generated in time course reactions of endonuclease V with dimer-containing form I DNA in the absence of NaCl, and in 25, 50, and 100 mM NaCl. The degree of processivity was evaluated in terms of the mass fraction of form III (linear) DNA which was produced as a function of the fraction of form I DNA remaining. Processivity is maximal in the absence of NaCl and decreases as the NaCl concentration is increased. At 100 mM NaCl, processivity is abolished and endonuclease V generates incisions in DNA at the site of dimers by a distributive reaction mechanism. The change from the distributive to a processive reaction mechanism occurs at NaCl concentrations slightly below 50 mM. The high degree of processivity which is observed in the absence of NaCl is reversible to the distributive mechanism, as demonstrated by experiments in which the NaCl concentration was increased during the time course reaction. In addition, unirradiated DNA inhibited the incision of irradiated DNA only at NaCl concentrations at which processivity was observed.
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PMID:The DNA scanning mechanism of T4 endonuclease V. Effect of NaCl concentration on processive nicking activity. 352 29

Electron microscopy of UV-irradiated circular DNA molecules which had been treated with T4 endonuclease V revealed the formation of multimeric DNA structures in addition to the expected conversion of the superhelical DNA molecules into nicked circular and linear forms. The multimeric DNA molecules could be distinguished in electron micrographs from catenated molecules which were present in the original DNA preparation by a combination of rotary and single angle heavy metal shadowing. The complexity and frequency of these structures increased with time of reaction with endonuclease V. Their formation, as well as the endonuclease activity of enzyme, was dependent on UV irradiation of the DNA, and the complexes could be disrupted by prior phenol extraction and ethanol precipitation. Preparations of endonuclease V estimated to be 98% pure by mass promoted the same complex formation between DNA molecules as did preparations estimated to be only 5-10% pure. In addition to these intermolecular structures, the formation of complexes between regions on the same DNA molecules was manifest as discrete double-stranded 'loops' 200-300 base pairs in length. DNA 'bubble structures' were also observed and may represent folding of the 'loops' onto adjacent segments of DNA. These results suggest that at least one active form of T4 endonuclease V may be a multimeric complex of enzyme molecules in association with DNA.
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PMID:T4 endonuclease V promotes the formation of multimeric DNA structures. 354 3

A comparison was made of the activity of the UV-specific endonucleases of bacteriophage T4 (T4 endonuclease V) and of Micrococcus luteus on ultravilet light-irradiated DNA substrates of defined sequence. The two enzymes cleave DNA at the site of pyrimidine dimers with the same frequency. The products of the cleavage reaction are the same, suggesting that the scission of DNA by T4 endonuclease V occurs via the combined actin of a pyrimidine dimer specific DNA glycosylase and an apyrimidinic-apurinic (AP) endonuclease as was recently shown for the M. luteus enzyme. The pyrimidine dimer DNA-glycosylase activity of both enzymes is more active on double-stranded DNA than it is on single-stranded DNA.
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PMID:Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus UV-specific endonucleases. 625 91

T4 endonuclease V [endodeoxyribonuclease (pyrimidine dimer), EC 3.1.25.1)], which is involved in repair of UV-damaged DNA, has been purified to apparent physical homogeneity. Incubation of UV-irradiated poly(dA).poly(dT) with the purified enzyme preparations resulted in production of alkali-labile apyrimidinic sites, followed by formation of nicks in the polymer. The activity to produce alkali-labile sites was optimal in a relatively broad pH range (pH 6.0-8.5), whereas the activity to form nicks had a narrow optimum near pH 6.5. By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks. The intermediate was used as substrate for the assay of apurinic/apyrimidinic DNA endonuclease activity [endodeoxyribonuclease (apurinic or apyrimidinic, EC 2.1.25.2]. The two activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme preparations that contained only a 16,000-dalton polypeptide. An enzyme fraction from cells infected with bacteriophage T4v1, a mutant that is sensitive to UV radiation, was defective in both glycosylase and endonuclease activities. Moreover, occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities, and suppression of the mutation rendered both activities partially active. These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single polypeptide chain coded by the denV gene of bacteriophage T4. Because the two activities exhibited different thermosensitivity, it was further suggested that conformation of the active sites for these activities may be different.
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PMID:Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA. 626 6

We performed experiments to determine whether the phage T4-induced UV endonuclease activity is a single protein containing both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities. The UV endonuclease activity is induced by the denV gene and codes for the glycosylase activity. We obtained several kinds of evidence that the protein containing the glycosylase activity also contains the apyrimidinic endonuclease activity. After chromatography on DEAE-cellulose, the two activities copurified during phosphocellulose chromatography and Sephadex G-100 chromatography, with a constant ratio of activities across the activity peaks. On Sephadex G-100 columns the molecular weights of the two activities agreed within 2,500 or less. When an extract of cells infected with the T4 V1 mutant was purified in exactly the same way as an extract of cells infected with T4 V1+, neither glycosylase nor apyrimidinic endonuclease activity was detected in the normal elution position of the T4 UV endonuclease activity. The glycosylase and apyrimidinic endonuclease activities were induced with similar kinetics, which were characteristic of immediate early rather than delayed early enzymes. This correlated well with the presumed major role of these activities in repairing thymine dimers in parental DNA before DNA replication begins. Finally, glycosylase and apyrimidinic endonuclease activities were lost in parallel during incubation of the enzyme at 46 degree C. Our results indicated that both of these enzyme activities are contained in the same enzyme molecule and, probably, in the same polypeptide.
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PMID:Evidence that the UV endonuclease activity induced by bacteriophage T4 contains both pyrimidine dimer-DNA glycosylase and apyrimidinic/apurinic endonuclease activities in the enzyme molecule. 627 Mar 74

Recent studies have shown purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phage T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV+ phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing apurinic or apyrimidinic sites to reactions containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity.
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PMID:den V gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities. 627 Mar 75

Pyrimidine dimer (PD)-DNA glycosylase activity has been reported in both the M. luteus and phage T4 UV endonucleases. In the present studies the T4 PD-DNA glycosylase has been purified close to physical homogeneity using an assay that measures the release of free thymine from UV-irradiated poly ([H5] dT):poly (dA), after the photo-reversal of thymine-thymine dimers. The activity has also been demonstrated in vivo following infection of UV-irradiated E. coli uvr- cells with phage T4. Under these conditions the T4 PD-DNA glycosylase accounts quantitatively for all thymine-containing PD excised from [3H] labeled E. coli DNA. In vitro the T4 PD-DNA glycosylase has an associated AP endonuclease activity that incises UV-irradiated DNA 3 to the apyrimidinic sites created by the glycosylase. However, the glycosylase/AP endonuclease reaction mechanism in vitro does not appear to be a concerted one. In addition, a T4 phage with a temperature-sensitive mutation in the denV gene shows wild-type levels of survival at the permissive temperature, despite the fact that in vitro, extracts of E. coli infected with this mutant show no detectable phage-coded AP endonuclease at 28 degrees C. Thus the exact role of the T4 AP endonuclease in the incision of UV-irradiated DNA dimer in vivo is not clear. The ratio of excised non-containing nucleotides to dimer-containing nucleotides following infection of UV-irradiated E. coli with phage T4 denV+ yields a calculated average repair patch size of approximately 7 nucleotides. In contrast, the calculated average patch size in uninfected E. coli is approximately 70 nucleotides. Thus the extent of excision/resynthesis of UV-irradiated DNA may be determined by the specific mode of incision of the DNA at PD. When uninfected E. coli (uvr+) is exposed to UV radiation, a fraction of the excised thymine-containing PD contain photolabile thymine, suggesting the presence of PD-DNA glycosylase in E. coli. The role of this putative activity in the metabolism of UV-irradiated DNA is under investigation.
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PMID:Pyrimidine dimer-DNA glycosylases: studies on bacteriophage T4-infected and on uninfected Escherichia coli. 675 48

The replication of the single-stranded DNA of parvovirus Minute-Virus-of-MIce (MVM) was depressed when virus was exposed to UV-light prior to infection of mouse fibroblasts. Most of the viral DNA containing pyrimidine dimers was permanently blocked in its conversion to double-stranded replicative forms (RF). Yet dimers might be tolerated to a low extent, considering that a minor fraction of parental RF molecules was sensitive to the action of the UV-specific endonuclease V of bacteriophage T4, UV-irradiation of the cells prior to infection with UV-damaged MVM increased the levels of both parental RF and total viral DNA synthesized. The sensitivity of parental RF molecules to the UV-specific endonuclease was little enhanced by preirradiation of the cells and did not appear to be sufficient to account for the stimulation of RF formation in those cultures. Since parvoviral single-stranded DNA is not a substrate for nucleotidyl excision repair, one interpretation of these results would be that the process(es) activated in preirradiated cells overcome(s) barriers to viral DNA replication other than elongation blocks at pyrimidine dimers. Alternatively, pyrimidine dimers tolerated in pretreated cultures might become protected from attack by the UV-endonuclease.
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PMID:Are pyrimidine dimers tolerated during DNA replication of UV-irradiated parvovirus minute-virus-of-mice in mouse fibroblasts? 681 34

Although Micrococcus luteus UV endonuclease has been reported to be an 18-kDa enzyme with possible homology to the 16-kDa endonuclease V from bacteriophage T4 (Gordon, L. K., and Haseltine, W. A. (1980) J. Biol. Chem. 255, 12047-12050; Grafstrom, R. H., Park, L., and Grossman, L. (1982) J. Biol. Chem. 257, 13465-13474), this study describes three independent purification schemes in which M. luteus UV damage-specific or pyrimidine dimer-specific nicking activity was associated with two proteins of apparent molecular masses of 31 and 32 kDa. An 18-kDa contaminant copurified with the doublet through many of the chromatographic steps, but it was determined to be a homolog of Escherichia coli ribosomal protein L6. Edman degradation analyses of the active proteins yielded identical NH2-terminal amino acid sequences. The corresponding gene (pdg, pyrimidine dimer glycosylase) was cloned. The protein bears strong sequence similarities to the E. coli repair proteins endonuclease III and MutY. Nonetheless, traditionally purified M. luteus protein acted exclusively on cis-syn thymine dimers; it was unable to cleave site-specific oligonucleotide substrates containing a trans-syn -I, (6-4), or Dewar thymine dimer, a 5,6-dihydrouracil lesion, or an A:G or A:C mismatch. The UV endonuclease incised cis-syn dimer-containing DNA in a dose-dependent manner and exhibited linear kinetics within that dose range. Enzyme activity was inhibited by the presence of NaCN or NaBH4 with NaBH4 additionally being able to trap a covalent enzyme-substrate product. These last findings confirm that the catalytic mechanism of M. luteus UV endonuclease, like those of other glycosylase/AP lyases, involves an imino intermediate.
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PMID:Purification and cloning of Micrococcus luteus ultraviolet endonuclease, an N-glycosylase/abasic lyase that proceeds via an imino enzyme-DNA intermediate. 755 10

Deinococcus radiodurans and other members of the same genus share extraordinary resistance to the lethal and mutagenic effects of ionizing and u.v. radiation and to many other agents that damage DNA. While it is known that this resistance is due to exceedingly efficient DNA repair, the molecular mechanisms responsible remain poorly understood. Following very high exposures to u.v. irradiation (e.g. 500 J m-2, which is non-lethal to D. radiodurans), this organism carries out extremely efficient excision repair accomplished by two separate nucleotide excision repair pathways acting simultaneously. One pathway requires the uvrA gene and appears similar to the UvrABC excinuclease pathway defined in Escherichia coli. The other excision repair pathway is specific for u.v. dimeric photoproducts, but is not mediated by a pyrimidine dimer DNA glycosylase. Instead, it is initiated by a second bona fide endonuclease that may recognize both pyrimidine dimers and pyrimidine-(6-4)pyrimidones. After high doses of ionizing-radiation (e.g. 1.5 Mrad), D. radiodurans can mend > 100 double-strand breaks (dsb) per chromosome without lethality or mutagenesis. Both dsb mending and survival are recA-dependent, indicating that efficient dsb mending proceeds via homologous recombination. D. radiodurans contains multiple chromosomes per cell, and it is proposed that dsb mending requires extensive recombination amongst these chromosomes, a novel phenomenon in bacteria. Thus, D. radiodurans may serve as an easily accessible model system for the double-strand-break-initiated interchromosomal recombination that occurs in eukaryotic cells during mitosis and meiosis.
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PMID:DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans. 798 97


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