Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.99.3 (PRE)
 1,923 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Survival and repair of DNA following ultraviolet (254-nm) radiation have been investigated in ICR 2A, a cultured cell line from haploid embryos of the grassfrog, Rana pipiens. Survival curves from cells recovering in the dark gave mean lethal dose value (Do) in the range 1.5--1.7 Jm-2 for both haploid and diploid cell stocks. The only significant difference observed between haploids and diploids was in the extent of the shoulder at low fluence (Dq), the value for exponentially multiplying diploid cells (3.0 Jm-2) being higher than that found for haploids (1.2 Jm-2). Irradiation of cultures reversibly blocked in the G1 phase of the cell cycle gave survival-curve coefficients indistinguishable between haploids and diploids. Post-irradiation exposure to visible light restored colony-forming capacity and removed chromatographically estimated pyrimidine dimers from DNA at the same rates. After fluences killing 90% of the cells, complete restoration of survival was obtained after 60-min exposure to 500 foot-candles, indicating that in this range lethality is entirely photoreversible and therefore attributable to pyrimidine dimers in DNA. Dimer removal required illumination following ultraviolet exposure, intact cells and physiological temperature, implying that the photoreversal involved DNA photolyase activity. Excision-repair capacity was slight, since no loss of dimers could be detected chromatographically during up to 48 h incubation in the dark and since autoradiographically detected "unscheduled DNA synthesis" was limited to a 2-fold increase saturated at 10 Jm-2. These properties make ICR 2A frog cells useful to explore how DNA-repair pathways influence mutant yield.
Mutat Res 1979 Sep
PMID:Survival and DNA repair in ultraviolet-irradiated haploid and diploid cultured frog cells. 31 17

The ultraviolet-light induction of DNA damage has been measured in the epidermis of hairless mice with the use of damage-specific endonucleases from Micrococcus luteus. The rates of induction of endonuclease-sensitive sites in HRS/J/Anl and Skh:hairless-1 mice were 6.1 +/- 0.5 X 10(-11) and 6.5 +/- 0.8 X 10(-11)/dalton/J/sq m from a FS40 fluorescent sun lamp (280 to 400 nm), respectively. Enzymatic photoreactivation with yeast photoreactivating enzyme showed that approximately 80% of the endonuclease-sensitive sites were cycloburyl pyrimidine dimers. In both strains of mice the pyrimidine dimers remained in high-molecular-weight DNA for 24 hr after irradiation. These data show that mouse epithelial cells in vivo have little or no capacity for the excision repair of pyrimidine dimers.
Cancer Res 1977 Sep
PMID:Induction and persistence of pyrimidine dimers in the epidermal DNA of two strains of hairless mice. 88 73

Photoreactivation of pyrimidine dimers in mammalian cells occurs under our experimental conditions but has not been observed under conditions used by others. We have tested three possible differences in experimental procedures including dimer separation and analysis methods, illumination conditions and cell culture techniques. We show that out methods of dimer separation and analysis indeed measure cis-syn pyrimidine dimers and give results in quantitative agreement with the methods of others. We find that while light pre-illumination of fibroblasts from the xeroderma pigmentosum line XP12BE or of normal cells does not affect the cellular capacity for dimer photoreactivation. However, we show that cell culture conditions can affect photoreactivating enzyme levels and thus, cellular dimer photoreactivation capacity. Cells grown in Eagle's minimal essential medium (supplemented with 15% fetal bovine serum) contain very low levels of photoreactivating enzyme and cannot photoreactivate dimers in their DNA; however, companion cultures maintained in Dulbecco's modified Eagle's minimal medium do contain photoreactivating enzyme and can photoreactivate cellular dimers.
Biochim Biophys Acta 1976 Sep 06
PMID:Culture conditions affect photoreactivating enzyme levels in human fibroblasts. 96 54

DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate. The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 (EdFADH2) matched its absorption spectrum after correction for the presence of a small amount of inactive 5-deazaFADox. The quantum yield for dimer repair with EdFADH2 (phi EdFADH2 = 0.110) was 6-fold lower than that observed with apoenzyme reconstituted with FADH2. Excited-state redox potential calculations indicate that 5-deazaFADH2 singlet is a better one-electron donor (E = -3.5 V) than FADH2 singlet (E = -2.7 V). Other studies indicate that the quantum yield for electron transfer from reduced flavin singlet to pyrimidine dimer (0.88) is unaffected when FADH2 is replaced by 5-deazaFADH2. Enhanced back electron transfer from pyrimidine dimer radical to flavin radical may account for the decreased quantum yield observed with EdFADH2 since, in the ground state, 5-deazaFADH. is a better oxidant than FADH.. The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 plus 5,10-CH(+)-H4folate (EPtedFADH2) matched the absorption spectrum determined for enzyme-bound 5-deazaFADH2, indicating that the pterin chromophore was inactive as a sensitizer. This differs from results obtained with native enzyme, where pterin acts as a sensitizer via efficient singlet-singlet energy transfer to FADH2. The quantum yield for dimer repair by 5-deazaFADH2 bound to EPtedFADH2 (phi EPtedFADH2 = 0.0318) was 28.9% of that observed for EdFADH2. Spectroscopic studies indicate that singlet-singlet energy transfer in EPtedFADH2 is very efficient but only occurs in the "wrong" direction, i.e., from excited 5-deazaFADH2 to pterin.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 1992 Sep 15
PMID:Effect of 5-deazaflavin on energy transduction during catalysis by Escherichia coli DNA photolyase. 139 Jun 27

Photolyases reverse the effects of UV light on cells by converting cyclobutane dipyrimidine photoproducts (pyrimidine dimers, Pyr mean value of Pyr) into pyrimidine monomers in a light-dependent reaction. Previous work has suggested that, based on substrate preference, there are two classes of photolyase: DNA photolyase as exemplified by the Escherichia coli enzyme, and RNA photolyases found in plants such as Nicotiana tabacum and Phaseolus vulgaris. In experiments aimed at identifying substrate determinants, including the pentose ring, for binding and catalysis by E. coli DNA photolyase we tested several Pyr mean value of Pyr. We found that the enzyme has relative affinities for photodimers of T mean value of T greater than or equal to U mean value of T greater than U mean value of U much greater than C mean value of C and that the E-FADH2 form of the enzyme repairs these dimers at 366 nm with absolute quantum yields of 0.9 (T mean value of T), 0.8 (U mean value of T), 0.6 (U mean value of U), and 0.05 (C mean value of C). The enzyme also repairs an isolated thymine dimer and the synthetic substrate, 1,1'-trimethylene-bis (thymine) cyclobutane dimer. Unexpectedly, we found that this enzyme, previously thought to be specific for DNA, repairs uracil cyclobutane dimers in poly(rU). The affinity of photolyase for a uracil dimer in RNA is about 10(4)-fold lower than that for a U mean value of U in DNA; however, once bound, the enzyme repairs the photodimer with the same quantum yield whether the dimer is in ribonucleoside or deoxyribonucleoside form.
Biochemistry 1991 Sep 03
PMID:Effect of base, pentose, and phosphodiester backbone structures on binding and repair of pyrimidine dimers by Escherichia coli DNA photolyase. 171 50

We have cloned the phr gene that encodes DNA photolyase from Salmonella typhimurium by in vivo complementation of Escherichia coli phr gene defect. The S.typhimurium phr gene is 1419 base pairs long and the deduced amino acid sequence has 80% identity with that of E. coli photolyase. We expressed the S.typhimurium phr gene in E.coli by ligating the E.coli trc promoter 5' to the gene, and purified the enzyme to near homogeneity. The apparent molecular weight of S.typhimurium photolyase is 54,000 dalton as determined by SDS-polyacrylamide gel electrophoresis, which is consistent with the calculated molecular weight of 53,932 dalton from the deduced phr gene product. S.typhimurium photolyase is purple-blue in color with near UV-visible absorption peaks at 384, 480, 580, and 625 nm and a fluorescence peak at 470 nm. From the characteristic absorption and fluorescence spectra and reconstitution experiments, S.typhimurium photolyase appears to contain flavin and methenyltetrahydrofolate as chromophore-cofactors as do the E.coli and yeast photolyases. Thus, S.typhimurium protein is the third folate class photolyase to be cloned and characterized to date. The binding constant of S.typhimurium photolyase to thymine dimer in DNA is kD = 1.6 x 10(-9) M, and the quantum yield of photorepair at 384 nm is 0.5.
Nucleic Acids Res 1991 Sep 25
PMID:Cloning, sequencing, expression and characterization of DNA photolyase from Salmonella typhimurium. 184 Jun 65

The PHR1 gene of Saccharomyces cerevisiae encodes a photolyase which repairs specifically and exclusively pyrimidine dimers, the most frequent lesions induced in DNA by far-UV radiation. We have asked whether expression of PHR1 is modulated in response to UV-induced DNA damage and to DNA-damaging agents that induce lesions structurally dissimilar to pyrimidine dimers. Using a PHR1-lacZ fusion gene in which expression of beta-galactosidase is regulated by PHR1 5' regulatory elements, we found that exposure of cells to 254-nm light, 4-nitroquinoline-N-oxide, methyl methanesulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine induced synthesis of increased amounts of fusion protein. In contrast to these DNA-damaging agents, neither heat shock nor exposure to photoreactivating light elicited a response. Induction by far-UV radiation was evident both when the fusion gene was carried on a multicopy plasmid and when it replaced the endogenous chromosomal copy of PHR1, and it was accompanied by an increase in the steady-state concentration of PHR1-lacZ mRNA. Northern (RNA) blot analysis of PHR1 mRNA encoded by the chromosomal locus was consistent with either enhanced transcription of PHR1 after DNA damage or stabilization of the transcripts. Neither the intact PHR1 or RAD2 gene was required for induction. Comparison of the region of PHR1 implicated in regulation of its expression with other damage-inducible genes from yeast cells revealed a common conserved sequence that is present in the PHR1, RAD2, and RNR2 genes and is required for damage inducibility of the latter two genes. These sequences may constitute elements of a damage-responsive regulon in S. cerevisiae.
Mol Cell Biol 1990 Sep
PMID:Expression of the yeast PHR1 gene is induced by DNA-damaging agents. 211

On the Rhizobium leguminosarum PRE sym plasmid, fixABC and a novel gene fixW were identified upstream of the regulatory gene nifA. The molecular masses of FixABC, 29, 44 and 50 kDa respectively, were estimated by polyacrylamide gel electrophoresis (PAGE) and of FixW, 25 kDa, by PAGE and nucleotide sequencing. Hybridization studies using bacteroid mRNA as a probe showed that fixABC is one operon which can be transcribed independently of fixW. Nucleotide sequencing revealed that both fixW and fixA are preceded by a nif consensus promoter. The fixA promoter partly overlaps the 3'-terminal coding region of fixW, indicating that readthrough from fixW into fixA is possible. Two open reading frames, ORF71 and ORF79, precede fixW and form one operon with fixW. ORF71 contains sequences homologous to the fixA promoter and 5'-terminal coding region. One more duplication of fixA sequences was detected, also located within the sym plasmid nif/fix clusters. One duplication of fixW sequences was found. No fixW homologue could be found in other nitrogen fixing organisms except in a number of R. leguminosarum strains.
Mol Gen Genet 1989 Sep
PMID:Characterization and nucleotide sequence of a novel gene fixW upstream of the fixABC operon in Rhizobium leguminosarum. 255 70

Lysogenic development by bacteriophage lambda is known to require the coordinate expression of two phage operons. Coordinate control is achieved by a positive regulatory mechanism which activates transcription from the promoters of these operons, PRE and PI. The positive effector is the phage regulatory protein cII. We now identify and characterize a third cII-dependent transcription unit on phage lambda, which is positioned in the middle of the Q regulatory gene and has an anti-Q orientation. We demonstrate the cII-dependent function of this promoter and precisely map its 5' transcription start-site both in vitro and in vivo. Most importantly, we show that cII binding and transcription activation at PaQ occur at essentially the same cII levels as those required for PRE and PI activation, and that all three promoters respond to cII at the same time following phage infection. In addition, DNase protection studies demonstrate that cII selectively interacts with the same TTGCN6TTGC DNA sequence repeat in the -35 region of PaQ which cII interacts with at both PRE and PI. We find that cII also binds other TTGCN6TTGC repeat sequences on lambda but binding at these sites does not lead to productive transcription. We conclude that PaQ functions in concert with PRE and PI to regulate the lysogenic growth response of phage lambda. We presume that the PaQ directed anti-sense Q RNA transcript functions to down-regulate the expression of the Q gene, which is needed for the expression of all phage late genes during lytic growth.
J Biol Chem 1985 Sep 25
PMID:Characterization of a third, cII-dependent, coordinately activated promoter on phage lambda involved in lysogenic development. 293 30

In this study, a novel approach to the analysis of DNA repair in Escherichia coli was employed which allowed the first direct determination of the mechanisms by which endogenous DNA repair enzymes encounter target sites in vivo. An in vivo plasmid DNA repair analysis was employed to discriminate between two possible mechanisms of target site location: a processive DNA scanning mechanism or a distributive random diffusion mechanism. The results demonstrate that photolyase acts by a distributive mechanism within E. coli. In contrast, UvrABC-initiated excision repair occurs by a limited processive DNA scanning mechanism. A majority of the dimer sites on a given plasmid molecule were repaired prior to the dissociation of the UvrABC complex. Furthermore, plasmid DNA repair catalyzed by the UvrABC complex occurs without a detectable accumulation of nicked plasmid intermediates despite the fact that the UvrABC complex generates dual incisions in the DNA at the site of a pyrimidine dimer. Therefore, the binding or assembly of the UvrABC complex on DNA at the site of a pyrimidine dimer represents the rate-limiting step in the overall process of UvrABC-initiated excision repair in vivo.
J Biol Chem 1988 Sep 05
PMID:Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. II. UvrABC-initiated excision repair and photolyase-catalyzed dimer monomerization. 304 28


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