Ultraviolet-B-induced inactivation of human OGG1, the repair enzyme for removal of 8-oxoguanine in DNA.

The OGG1 proteins are DNA N-glycosylases-apurinic-apyrimidinic lyases that are responsible for the removal of 8-oxo-7,8-dihydroguanine (8-oxoG) base in DNA. The human enzyme (hOGG1) is a monomer of 345 amino acids containing 10 buried tryptophan (Trp) residues that are very sensitive to UVB irradiation. The photolysis quantum yield of these Trp residues is about 0.3 and 0.1 in argon- and air-saturated solutions, respectively. Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry shows that several cleavage sites are identical under aerobic and anaerobic photolysis of Trp residues; one of them includes the active site. Western blots and polyacrylamide gel electrophoresis indicate that fragments of high molecular size are also formed. In addition to common photochemical paths with argon-saturated solutions, specific reactions occur in air-saturated solutions of hOGG1. The photolysis rate is inhibited by more than 50% on binding of hOGG1 to a 34mer oligonucleotide containing a single 8-oxoG-C base pair. Binding to the oligonucleotide with 8-oxoG-C induced a 20% quenching of the hOGG1 fluorescence, suggesting interaction of nucleic acid bases with the Trp residue(s) responsible for the photolysis. Using 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-FapyG) and 8-oxoG as substrates, it is shown that protein photolysis induces photoinactivation of the DNA N-glycosylase activities. The excision of 8-oxoG is more affected than that of Me-FapyG at the same dose of UVB irradiation under both air and argon conditions. Besides the role of Trp residues, the possible involvement of Cys 253 in the photoinactivation process of hOGG1 is discussed.

Synergism between base excision repair, mediated by the DNA glycosylases Ntg1 and Ntg2, and nucleotide excision repair in the removal of oxidatively damaged DNA bases in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, inactivation of the two DNA N-glycosylases Ntg1p and Ntg2p does not result in a spontaneous mutator phenotype, whereas simultaneous inactivation of Ntglp, Ntg2p and Radlp or Rad14p, both of which are involved in nucleotide excision repair (NER), does. The triple mutants rad1 ntg1 ntg2 and rad14 ntg1 ntg2 show 15- and 22-fold increases, respectively, in spontaneous forward mutation to canavanine resistance (CanR) relative to the wild-type strain (WT). In contrast, neither of these triple mutants shows an increase in the incidence of Lys+ revertants of the lys1-1 ochre allele. Furthermore, the rad1 ntg1 ntg2 mutant is hypersensitive to the lethal effect of H2O2 relative to WT, rad1 and ntg1 ntg2 mutant strains. Moreover, the rad1 ntg1 ntg2 strain is hypermutable (CanR and Lys+) upon exposure to H2O2, relative to WT, rad1 and ntg1 ntg2 strains. Mutagen sensitivity and enhanced mutagenesis in the rad1 ntg1 ntg2 triple mutant, relative to the other strains tested, were also observed upon exposure to oxidizing agents such as tertbutylhydroperoxide and menadione. In contrast, the sensitivity of the rad1 ntg1 ntg2 triple mutant to gamma-irradiation does not differ from that of the WT. However, the triple mutant shows an increase in the frequency of Lys+ revertants recovered after gamma-irradiation. The results reported in this study demonstrate that base excision repair (BER) mediated by Ntglp and Ntg2p acts synergistically with NER to repair endogenous or induced lethal and mutagenic oxidative DNA damage in yeast. The substrate specificity of Ntg1 p and Ntg2p, and the spectrum of lesions induced by the DNA-damaging agents used, strongly suggest that oxidized DNA bases, presumably oxidized pyrimidines, represent the major targets of this repair pathway.

Catalytic and DNA binding properties of the ogg1 protein of Saccharomyces cerevisiae: comparison between the wild type and the K241R and K241Q active-site mutant proteins.

The Ogg1 protein of Saccharomyces cerevisiae belongs to a family of DNA glycosylases and apurinic/apyrimidinic site (AP) lyases, the signature of which is the alpha-helix-hairpin-alpha-helix-Gly/Pro-Asp (HhH-GPD) active site motif together with a conserved catalytic lysine residue, to which we refer as the HhH-GPD/K family. In the yeast Ogg1 protein, yOgg1, the HhH-GPD/K motif spans residues 225-260 and the conserved lysine is K241. In this study, we have purified the K241R and K241Q mutant proteins and compared their catalytic and DNA binding properties to that of the wild-type yOgg1. The results show that the K241R mutation greatly impairs both the DNA glycosylase and the AP lyase activities of yOgg1. Specificity constants for cleavage of a 34mer oligodeoxyribonucleotide containing a 7,8-dihydro-8-oxoguanine (8-OxoG) paired with a cytosine, [8-OxoG.C], are 56 x 10(-)(3) and 5 x 10(-)(3) min(-)(1) nM(-)(1) for the wild-type and the K241R protein, respectively. On the other hand, the K241Q mutation abolishes the DNA glycosylase and AP lyase activities of yOgg1. In contrast, the K241R and K241Q proteins have conserved wild-type DNA binding properties. K(dapp) values for binding of [8-OxoG.C] are 6.9, 7.4, and 4.8 nM for the wild-type, K241R, and K241Q proteins, respectively. The results also show that AP site analogues such as 1, 3-propanediol (Pr), tetrahydrofuran (F), or cyclopentanol (Cy) are not substrates but constitute good inhibitors of the wild-type yOgg1. Therefore, we have used a 59mer [Pr.C] duplex to further analyze the DNA binding properties of the wild-type, K241R, and K241Q proteins. Hydroxyl radical footprints of the wild-type yOgg1 show strong protection of six nucleotides centered around the Pr lesion in the damaged strand. On the complementary strand, only the cytosine placed opposite Pr was strongly protected. The same footprints were observed with the K241R and K241Q proteins, confirming their wild-type DNA binding properties. These results indicate that the K241Q mutant protein can be used to study interactions between yOgg1 and DNA containing metabolizable substrates such as 8-OxoG or an AP site.

The effect of single engine fixed wing air transport on rate-responsive pacemakers.

BACKGROUND: Insufficient information exists about the safety of patients with accelerometer-based rate-responsive pacemakers in air transport by general aviation aircraft. METHODS: The response in pacing rate of two types of accelerometer-based rate-responsive pacemakers with data logging capabilities was studied during test flights with single engine fixed wing aircraft. Results were compared with the rate-response of these pacemakers during transportation by car and were also interpreted in respect to physiological heart rate response of aircrew during flights in single engine fixed wing aircraft. In addition, a continuous accelerometer readout was recorded during a turbulent phase of flight. This recording was used for a pacemaker-simulator experiment with maximal sensitive motion-sensor settings. RESULTS: Only a minor increase in pacing rate due to aircraft motion could be demonstrated during all phases of flight at all altitudes with the pacemakers programmed in the normal mode. This increase was of the same magnitude as induced during transport by car and would be of negligible influence on the performance of the individual pacemaker patient equipped with such a pacemaker. Moreover, simultaneous Holter monitoring of the pilots during these flights showed a similar rate-response in natural heart rate compared with the increase in pacing rate induced by aircraft motion in accelerometer-based rate-responsive pacemakers. No sensor-mediated pacemaker tachycardia was seen during any of these recordings. However, a 15% increase in pacing rate was induced by severe air turbulence. Programming the maximal sensitivity of the motion sensor into the pacemaker could, on the other hand, induce a significant increase in pacing rate as was demonstrated by the simulation experiments. CONCLUSION: These results seem to rule out potentially dangerous or adverse effects from motional or vibrational influences during transport in single engine fixed wing aircraft on accelerometer-based rate-responsive pacemakers with normal activity sensor settings.

Substrate specificities of the ntg1 and ntg2 proteins of Saccharomyces cerevisiae for oxidized DNA bases are not identical.

Two genes of Saccharomyces cerevisiae, NTG1 and NTG2, encode proteins with a significant sequence homology to the endonuclease III of Escherichia coli. The Ntg1 and Ntg2 proteins were overexpressed in E.coli and purified to apparent homogeneity. The substrate specificity of Ntg1 and Ntg2 proteins for modified bases in oxidatively damaged DNA was investigated using gas chromatography/isotope-dilution mass spectrometry. The substrate used was calf-thymus DNA exposed to gamma-radiation in N2O-saturated aqueous solution. The results reveal excision by Ntg1 and Ntg2 proteins of six pyrimidine-derived lesions, 5-hydroxy-6-hydrothymine, 5-hydroxy-6-hydrouracil, 5-hydroxy-5-methylhydantoin, 5-hydroxyuracil, 5-hydroxycytosine and thymine glycol, and two purine-derived lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine from gamma-irradiated DNA. In contrast, Ntg1 and Ntg2 proteins do not release 8-hydroxyguanine or 8-hydroxyadenine from gamma-irradiated DNA. The Ntg1 and Ntg2 proteins also release 2, 6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine from damaged poly(dG-dC).poly(dG-dC). Excision was measured as a function of enzyme concentration and time. Furthermore, kinetic parameters were determined for each lesion. The results show that kinetic constants varied among the different lesions for the same enzyme. We also investigated the capacity of the Ntg1 and Ntg2 proteins to cleave 34mer DNA duplexes containing a single 8-OH-Gua residue mispaired with each of the four DNA bases. The results show that the Ntg1 protein preferentially cleaves a DNA duplex containing 8-OH-Gua mispaired with a guanine. Moreover, the Ntg1 protein releases free 8-OH-Gua from 8-OH-Gua/Gua duplex but not from duplexes containing 8-OH-Gua mispaired with adenine, thymine or cytosine. In contrast, the Ntg2 protein does not incise duplexes containing 8-OH-Gua mispaired with any of the four DNA bases. These results demonstrate that substrate specificities of the Ntg1 and Ntg2 proteins are similar but not identical and clearly different from that of the endonuclease III of E.coli and its homologues in Schizosaccharomyces pombe or human cells.

Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine.

A spontaneous mutator strain of Escherichia coli (fpg mutY) was used to clone the OGG1 gene of Saccharomyces cerevisiae, which encodes a DNA glycosylase activity that excises 7,8-dihydro-8-oxoguanine (8-OxoG). E. coli (fpg mutY) was transformed by a yeast DNA library, and clones that showed a reduced spontaneous mutagenesis were selected. The antimutator activity was associated with pYSB10, an 11-kbp recombinant plasmid. Cell-free extracts of E. coli (fpg mutY) harboring pYSB10 possess an enzymatic activity that cleaves a 34-mer oligonucleotide containing a single 8-oxoG opposite a cytosine (8-OxoG/C). The yeast DNA fragment of 1.7 kbp that suppresses spontaneous mutagenesis and overproduces the 8-OxoG/C cleavage activity was sequenced and mapped to chromosome XIII. DNA sequencing identified an open reading frame, designated OGG1, which encodes a protein of 376 amino acids with a molecular mass of 43 kDa. The OGG1 gene was inserted in plasmid pUC19, yielding pYSB110. E. coli (fpg) harboring pYSB110 was used to purify the Ogg1 protein of S. cerevisiae to apparent homogeneity. The Ogg1 protein possesses a DNA glycosylase activity that releases 8-OxoG and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. The Ogg1 protein preferentially incises DNA that contains 8-OxoG opposite cytosine (8-OxoG/C) or thymine (8-OxoG/T). In contrast, Ogg1 protein does not incise the duplex where an adenine is placed opposite 8-OxoG (8-OxoG/A). The mechanism of strand cleavage by Ogg1 protein is probably due to the excision of 8-OxoG followed by a beta-elimination at the resulting apurinic/apyrimidinic site.

The chronotropic response of the sinus node to exercise: a new method of analysis and a study of pacemaker patients.

In this study, the intercepts and slopes of the regression lines of sinus heart rate response to exercise were examined as a possible means of assessing normal and abnormal chronotropic responses. The regressions were plotted for 223 normal subjects and 93 pacemaker patients during progressive bicycle exercise. The 93 pacemaker patients consisted of two groups, (a) 46 with symptomatic sick sinus syndrome (daytime sinus or nodal bradycardia of < 45.min-1 or atrial asystole > 1.5 s together with other features of the syndrome) and (b) 47 with second- or third-degree AV block (in whom a one-to-one AV relationship was either restored by dual chamber pacing or in whom normal AV conduction was present at the time of exercise). Examination of the regression lines showed them to be sufficiently linear in both normal subjects and pacemaker patients to allow calculation of the intercept and slope values. Multiple regression analysis of these values showed that the AV block group did not differ significantly from the normal individuals. However, in the patients with sick sinus syndrome, the intercept but not the slope values were significantly lower than in the normal subjects (P < 0.01). The patients with sick sinus syndrome appeared to have an abnormality of sinus node function, where the basic rate setting is too low at all levels of exercise, although the acceleration with exercise is normal. Among the sick sinus syndrome patients, only four (9%) had intercept values lying more than 2 standard deviations below that of the normal control group and were deemed to be chronotropically incompetent. Finally, the problems with the various methods used to investigate chronotropic incompetence are reviewed.(ABSTRACT TRUNCATED AT 250 WORDS)

Formamidopyrimidine DNA glycosylase in the yeast Saccharomyces cerevisiae.

A DNA glycosylase that excises, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) from double stranded DNA has been purified 28,570-fold from the yeast Saccharomyces cerevisiae. Gel filtration chromatography shows that yeast Fapy DNA glycosylase has a molecular weight of about 40 kDa. The Fapy DNA glycosylase is active in the presence of EDTA, but is completely inhibited by 0.2 M KCl. Yeast Fapy DNA glycosylase does not excise N7-methylguanine, N3-methyladenine or uracil. A repair enzyme for 7,8-dihydro-8-oxoguanine (8-OxoG) co-purifies with the Fapy DNA glycosylase. This repair activity causes strand cleavage at the site of 8-OxoG in DNA duplexes. The highest rate of incision of the 8-OxoG-containing strand was observed for duplexes where 8-OxoG was opposite guanine. The mode of incision at 8-OxoG was not established yet. The results however suggest that the Fapy- and 8-OxoG-repair activities are associated with a single protein.

[Xanthine oxidase activity: NAD+-dependent and O2-dependent forms in carrageenan granuloma in the rat]. / Activité de la xanthine oxydase: formes nad+-dépendante et o2-dépendante dans le granulome à la carragénine chez le rat.

Xanthine oxidase activity: NAD+-dependent form (D) and O2-dependent form (O) were carried out in cytosol supernatants of connective tissue growth (T.C.N.F.), skin tail, liver and plasma of carrageenan induced granuloma in the Rat. The specific activities of skin, liver and plasma were normal in animals with a granuloma. The total specific activity (D + O): 7.53 +/- 0.98 mU/mg protein, and the percentage of form O: 51.6 +/- 5.1 of the granulomatous tissue as compared to the tail are significantly increased. These results suggest the likely function of xanthine oxidase during the inflammatory response.

[Xanthine oxidase activity: O2-dependent and NAD+-dependent forms in the liver, in rats with adjuvant arthritis and hepatitis]. / Activité de la xanthine oxydase: formes O2-dépendante et NAD+-dépendante du foie, chez le rat atteint d'arthrite et d'hépatite à adjuvant.

Xanthine oxidase activity: O2-dependent and NAD+-dependent forms, were carried out in cytosol supernatant of Rat liver homogenat with adjuvant and hepatocytes induced arthritis and hepatitis. Both forms were increased without modification of their ratio. These results suggest that xanthine oxidase was implicated in the inflammatory reaction.