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M Dizdaroglu

Publications and source records attributed to M Dizdaroglu.

At least 19 recordsLinked to original sources
PubMed2001

Substrate specificity and excision kinetics of Escherichia coli endonuclease VIII (Nei) for modified bases in DNA damaged by free radicals.

Endonuclease VIII (Nei) is one of three enzymes in Escherichia coli that are involved in base-excision repair of oxidative damage to DNA. We investigated the substrate specificity and excision kinetics of this DNA glycosylase for bases in DNA that have been damaged by free radicals. Two different DNA substrates were prepared by gamma-irradiation of DNA solutions under N(2)O or air, such that they contained a multiplicity of modified bases. Although previous studies on the substrate specificity of Nei had demonstrated activity on several pyrimidine derivatives, this present study demonstrates excision of additional pyrimidine derivatives and a purine-derived lesion, 4,6-diamino-5-formamidopyrimidine, from DNA containing multiple modified bases. Excision was dependent on enzyme concentration, incubation time, and substrate concentration, and followed Michaelis-Menten kinetics. The kinetic parameters also depended on the identity of the individual modified base being removed. Substrates and excision kinetics of Nei and a naturally arising mutant form involving Leu-90-->Ser (L90S-Nei) were compared to those of Escherichia coli endonuclease III (Nth), which had previously been determined under experimental conditions similar to those in this study. This comparison showed that Nei and Nth significantly differ from each other in terms of excision rates, although they have common substrates. The present work extends the substrate specificity of Nei and shows the effect of a single mutation in the nei gene on the specificity of Nei.

DNA Damage↗
PubMed2001

The Cockayne Syndrome group B gene product is involved in general genome base excision repair of 8-hydroxyguanine in DNA.

Cockayne Syndrome (CS) is a human genetic disorder with two complementation groups, CS-A and CS-B. The CSB gene product is involved in transcription-coupled repair of DNA damage but may participate in other pathways of DNA metabolism. The present study investigated the role of different conserved helicase motifs of CSB in base excision repair. Stably transformed human cell lines with site-directed CSB mutations in different motifs within its putative helicase domain were established. We find that CSB null and helicase motif V and VI mutants had greater sensitivity than wild type cells to gamma-radiation. Whole cell extracts from CSB null and motif V/VI mutants had lower activity of 8-hydroxyguanine incision in DNA than wild type cells. Also, 8-hydroxyguanine accumulated more in CSB null and motif VI mutant cells than in wild type cells after exposure to gamma-radiation. We conclude that a deficiency in general genome base excision repair of selective modified DNA base(s) might contribute to CS pathogenesis. Furthermore, whereas the disruption of helicase motifs V or VI results in a CSB phenotype, mutations in other helicase motifs do not cause this effect. The biological functions of CSB in different DNA repair pathways may be mediated by distinct functional motifs of the protein.

Amino Acid Sequence↗
PubMed2001

Effect of single mutations on the specificity of Escherichia coli FPG protein for excision of purine lesions from DNA damaged by free radicals.

The formamidopyrimidine N-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that is specific for the removal of purine-derived lesions from DNA damaged by free radicals and other oxidative processes. We investigated the effect of single mutations on the specificity of this enzyme for three purine-derived lesions in DNA damaged by free radicals. These damaging agents generate a multiplicity of base products in DNA, with the yields depending on the damaging agent. Wild type Fpg protein (wt-Fpg) removes 8-hydroxyguanine (8-OH-Gua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from damaged DNA with similar specificities. We generated five mutant forms of this enzyme with mutations involving Lys-57-->Gly (FpgK57G), Lys-57-->Arg (FpgK57R), Lys-155-->Ala (FpgK155A), Pro-2-->Gly (FpgP2G), and Pro-2-->Glu (FpgP2E), and purified them to homogeneity. FpgK57G and FpgK57R were functional for removal of FapyAde and FapyGua with a reduced activity when compared with wt-Fpg. The removal of 8-OH-Gua was different in that the specificity of FpgK57G was significantly lower for its removal from irradiated DNA, whereas wt-Fpg, FpgK57G, and FpgK57R excised 8-OH-Gua from H2O2/Fe(III)-EDTA/ascorbic acid-treated DNA with almost the same specificity. FpgK155A and FpgP2G had very low activity and FpgP2E exhibited no activity at all. Michaelis-Menten kinetics of excision was measured and kinetic constants were obtained. The results indicate an important role of Lys-57 residue in the activity of Fpg protein for 8-OH-Gua, but a lesser significant role for formamidopyrimidines. Mutations involving Lys-155 and Pro-2 had a dramatic effect with Pro-2-->Glu leading to complete loss of activity, indicating a significant role of these residues. The results show that point mutations significantly change the specificity of Fpg protein and suggest that point mutations are also expected to change specificities of other DNA repair enzymes.

Ascorbic Acid↗
PubMed2001

Measurement of 8-hydroxy-2'-deoxyadenosine in DNA by liquid chromatography/mass spectrometry.

8-Hydroxyadenine (8-OH-Ade) is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the C-8 position of adenine followed by oxidation. We describe the measurement of the nucleoside form of this compound, 8-hydroxy-2'-deoxyadenosine (8-OH-dAdo) in DNA by liquid chromatography/mass spectrometry (LC/MS). The developed methodology enabled the separation by LC of 8-OH-dAdo from intact and modified nucleosides in enzymic hydrolysates of DNA. Measurements by MS were performed using atmospheric pressure ionization-electrospray process. Isotope-dilution MS was applied for quantification using a stable isotope-labeled analog of 8-OH-dAdo. The level of sensitivity of LC/MS with selected-ion monitoring (SIM) for 8-OH-dAdo amounted to approximately 10 femtomol of this compound on the LC column. This level of sensitivity is similar to that previously reported using LC-tandem MS (LC/MS/MS) with multiple-reaction monitoring mode (MRM) (7.5 femtomol). This compound was quantified in DNA at a level of approximately one molecule/10(6) DNA bases using amounts of DNA as low as 5 microg. The results suggested that this lesion may be quantified in DNA at even lower levels, when more DNA is used for analysis. In addition, gas chromatography/isotope-dilution mass spectrometry with SIM (GC/IDMS-SIM) was applied to measure 8-OH-Ade in DNA following its removal from DNA by acidic hydrolysis. The background levels of 8-OH-dAdo and 8-OH-Ade measured by LC/IDMS-SIM and GC/IDMS-SIM, respectively, were nearly identical. In addition, DNA samples, which were exposed to ionizing radiation at different radiation doses, were analyzed by these techniques. Nearly identical results were obtained, indicating that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results. The level of sensitivity of GC/MS-SIM for 8-OH-Ade was also measured and found to be significantly greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS-MRM for 8-OH-dAdo. The results show that the LC/MS technique is well suited for the measurement of 8-OH-dAdo in DNA.

Animals↗
PubMed2001

Identification and quantification of 8,5'-cyclo-2'-deoxy-adenosine in DNA by liquid chromatography/ mass spectrometry.

Recent studies suggested that 8,5'-cyclo-2'-deoxyadenosine may play a role in diseases with defective nucleotide-excision repair. This compound is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the sugar moiety of 2'-deoxyadenosine. It is likely to be repaired by nucleotide-excision repair rather than by base-excision repair because of a covalent bond between the sugar and base moieties. We studied the measurement of 8,5'-cyclo-2'-deoxyadenosine in DNA by liquid chromatography/isotope-dilution mass spectrometry. A methodology was developed for the analysis of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography in DNA hydrolyzed to nucleosides by a combination of four enzymes, i.e., DNase I, phosphodiesterases I and II, and alkaline phosphatase. Detection by mass spectrometry was performed using atmospheric pressure ionization-electrospray process in the positive ionization mode. Results showed that liquid chromatography/isotope-dilution mass spectrometry is well suited for identification and quantification of 8,5'-cyclo-2'-deoxyadenosine in DNA. Both (5'R)- and (5'S)-diastereomers of 8,5'-cyclo-2'-deoxyadenosine were detected. The level of sensitivity of liquid chromatography/mass spectrometry with selected-ion monitoring amounted to 2 fmol of this compound on the column. The yield of 8,5'-cyclo-2'-deoxyadenosine was measured in DNA in aqueous solution exposed to ionizing radiation at doses from 2.5 to 80 Gray. Gas chromatography/mass spectrometry was also used to measure this compound in DNA. Both techniques yielded similar results. The yield of 8,5'-cyclo-2'-deoxyadenosine was comparable to the yields of some of the other major modified bases in DNA, which were measured using gas chromatography/mass spectrometry. The measurement of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography/mass spectrometry may contribute to the understanding of its biological properties and its role in diseases with defective nucleotide-excision repair.

Alkaline Phosphatase↗
PubMed2001

Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry.

Measurement of 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10(6) DNA bases can be detected using amounts of DNA as low as 2 microgram. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10(6) DNA bases, or even lower, when more DNA is used. Up to 50 microgram of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Repair of oxidative DNA damage in Drosophila melanogaster: identification and characterization of dOgg1, a second DNA glycosylase activity for 8-hydroxyguanine and formamidopyrimidines.

In Drosophila, the S3 ribosomal protein has been shown to act as a DNA glycosylase/AP lyase capable of releasing 8-hydroxyguanine (8-OH-Gua) in damaged DNA. Here we describe a second Drosophila protein (dOgg1) with 8-OH-Gua and abasic (AP) site DNA repair activities. The Drosophila OGG1 gene codes for a protein of 327 amino acids, which shows 33 and 37% identity with the yeast and human Ogg1 proteins, respectively. The DNA glycosylase activity of purified dOgg1 was investigated using gamma-irradiated DNA and gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The dOgg1 protein excises 8-OH-Gua and 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from gamma-irradiated DNA. with k(ca)(t)/K:(M) values of 21.0 x 10(-5) and 11.2 x 10(-5) (min(-1) nM(-1)), respectively. Enzymatic assays using oligodeoxyribonucleotides containing a single lesion show that dOgg1 displays a marked preference for DNA duplexes containing 8-OH-Gua, 8-OH-Ade or an AP site placed opposite a cytosine. The cleavage of the 8-OH-Gua-containing strand results from the excision of the damaged base followed by a ss-elimination reaction at the 3'-side of the resulting AP site. Cleavage of 8-OH-Gua.C duplex involves the formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydre. dOgg1 complements the mutator phenotype of fpg mutY mutants of Escherichia coli. Whole-mount in situ hybridizations on tissues at different stages of Drosophila development reveal that the dOGG1 messenger is expressed uniformly at a low level in cells in which mitotic division occurs. Therefore, Drosophila possesses two DNA glycosylase activities that can excise 8-OH-Gua and formamidopyrimidines from DNA, dOgg1 and the ribosomal protein S3.

Amino Acid Sequence↗
PubMed2000

Characterization of a novel 8-oxoguanine-DNA glycosylase activity in Escherichia coli and identification of the enzyme as endonuclease VIII.

8-Oxoguanine (G*), induced by reactive oxygen species, is mutagenic because it mispairs with A. The major G*-DNA glycosylase (OGG), namely, OGG1 in eukaryotes, or MutM in Escherichia coli, excises G* when paired in DNA with C, G, and T, but not A, presumably because removal of G* from a G*.A pair would be mutagenic. However, repair of G* will prevent mutation when it is incorporated in the nascent strand opposite A. This could be carried out by a second OGG, OGG2, identified in yeast and human cells. We have characterized a new OGG activity in E. coli and then identified it to be endonuclease VIII (Nei), discovered as a damaged pyrimidine-specific DNA glycosylase. Nei shares sequence homology and reaction mechanism with MutM and is similar to human OGG2 in being able to excise G* when paired with A (or G). Kinetic analysis of wild type Nei showed that it has significant activity for excising G* relative to dihydrouracil. The presence of OGG2 type enzyme in both E. coli and eukaryotes, which is at least as efficient in excising G* from a G*.A (or G) pair as from a G*.C pair, supports the possibility of G* repair in the nascent DNA strand.

DNA Damage↗
PubMed2000

Comparison of the levels of 8-hydroxyguanine in DNA as measured by gas chromatography mass spectrometry following hydrolysis of DNA by Escherichia coli Fpg protein or formic acid.

8-hydroxyguanine (8-OH-Gua) is one of many lesions generated in DNA by oxidative processes including free radicals. It is the most extensively investigated lesion, due to its miscoding properties and its potential role in mutagenesis, carcinogenesis and aging, and also to the existence of analytical methods using HPLC and gas chromatography mass spectrometry (GC/MS). Some studies raised the possibility of artifacts generated during sample preparation. We investigated several experimental conditions in order to eliminate possible artifacts during the measurement of 8-OH-Gua by GC/MS. Derivatization has been reported to produce artifacts by oxidation of guanine to 8-OH-Gua in acid-hydrolysates of DNA, although the extent of artifacts seems to depend on experimental conditions. For removal of 8-OH-Gua from DNA, we used either formic acid hydrolysis or specific enzymatic hydrolysis with Escherichia coli Fpg protein. Derivatization of enzyme-hydrolysates should not generate additional 8-OH-Gua because of the absence of guanine, which is not released by the enzyme, whereas guanine released by acid may be oxidized to yield 8-OH-Gua. The measurement of 8-OH-Gua in calf thymus DNA by GC/isotope-dilution MS (GC/IDMS) using these two different hydrolyses yielded similar levels of 8-OH-Gua. This indicated that no artifacts occurred during derivatization of acid-hydrolysates of DNA. Pyridine instead of acetonitrile and room temperature were used during derivatization. Pyridine reduced the level of 8-OH-Gua, when compared with acetonitrile, indicating its potential to prevent oxidation. Two different stable-isotope labeled analogs of 8-OH-Gua used as internal standards for GC/IDMS analysis yielded similar results. A comparison of the present results with the results of recent trials by the European Standards Committee for Oxidative DNA Damage (ESCODD) is also presented.

Animals↗
PubMed2000

Effect of single mutations in the OGG1 gene found in human tumors on the substrate specificity of the Ogg1 protein.

We have investigated the effect of single amino acid substitutions of conserved arginines on the catalytic activities of the human Ogg1 protein (alpha-hOgg1-Ser(326)) (wild-type alpha-hOgg1). Mutant forms of hOgg1 with mutations Arg(46)-->Gln (alpha-hOgg1-Gln(46)) and Arg(154)-->His (alpha-hOgg1-His(154)) have previously been identified in human tumors. The mutant proteins alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) were expressed in Escherichia coli and purified to homogeneity. The substrate specificities of these proteins and wild-type alpha-hOgg1 were investigated using gamma-irradiated DNA and the technique of gas chromatography/isotope-dilution mass spectrometry. All three enzymes excised 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) from gamma-irradiated DNA containing a multiplicity of base lesions. Michaelis-Menten kinetics of excision were measured. Significant differences between excision kinetics of these three enzymes were observed. Excision of FapyGua and 8-OH-Gua by wild-type alpha-hOgg1 was greater than that by alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154). The latter mutant protein was less active than the former. The diminished activity of the mutant proteins was more pronounced for 8-OH-Gua than for FapyGua. Cleavage assays were also performed using (32)P-labeled 34mer oligonucleotide duplexes containing a single 8-OH-Gua paired to each of the four DNA bases. The results obtained with the oligonucleotide containing the 8-OH-Gua/Cyt pair were in good agreement with those observed with gamma-irradiated DNA. Wild-type alpha-hOgg1 and its mutants repaired the three mismatches less efficiently than the 8-OH-Gua/Cyt pair. The substitution of Arg(154), in addition to diminishing the activity on 8-OH-Gua, relaxes the selectivity found in the wild-type alpha-hOgg1 for the base opposite 8-OH-Gua. Taken together the results show that the mutant forms alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) found in human tumors are defective in their catalytic capacities.

DNA↗
PubMed2000

Assignment of enzyme substrate specificity by principal component analysis of aligned protein sequences: an experimental test using DNA glycosylase homologs.

We have studied the relationship between amino acid sequence and substrate specificity in a DNA glycosylase family by characterizing experimentally the specificity of four new members of the family. We show that principal component analysis (PCA) of the sequence family correctly predicts the substrate specificity of one of the novel homologs even though conventional sequence analysis methods fail to group this homolog with other sequences of the same specificity. PCA also suggested, correctly, that another homolog characterized previously differs in its specificity from those sequences with which it clusters by conventional criteria. These results suggest that principal component analysis of sequence families can be a useful tool in annotating genome sequences when there is ambiguity concerning which subfamily a new homolog belongs to. Published 2000 Wiley-Liss, Inc.

Amino Acid Sequence↗
PubMed2000

Novel substrates of Escherichia coli nth protein and its kinetics for excision of modified bases from DNA damaged by free radicals.

Escherichia coli Nth protein (endonuclease III) is a DNA glycosylase with a broad substrate specificity for pyrimidine derivatives. We discovered novel substrates of E. coli Nth protein using gas chromatography/isotope-dilution mass spectrometry and DNA samples, which were damaged by gamma-irradiation or by H(2)O(2)/Fe(III)-EDTA/ascorbic acid. These were 4, 6-diamino-5-formamidopyrimidine, 5,6-dihydroxyuracil, and 5, 6-dihydroxycytosine. The first compound was recognized for the first time as a purine-derived substrate of the enzyme. We also investigated kinetics of excision of a multitude of modified bases from three damaged DNA substrates. Excision of modified bases was determined as a function of enzyme concentration, incubation time, and substrate concentration. Excision followed Michaelis-Menten kinetics. Kinetic parameters were determined for the following modified bases: 4,6-diamino-5-formamidopyrimidine, cis- and trans-thymine glycols, 5-hydroxycytosine, cis- and trans-uracil glycols, 5-hydroxyuracil, 5-hydroxy-5-methylhydantoin, alloxan, 5, 6-dihydroxycytosine, 5,6-dihydroxyuracil, 5-hydroxy-6-hydrothymine, and 5-hydroxy-6-hydrouracil. The results show that three newly discovered substrates were excised by the enzyme with a preference similar to excision of its known major substrates such as thymine glycol and 5-hydroxycytosine. Excision kinetics significantly depended on the nature of the damaged DNA substrates in agreement with previous results on other DNA glycosylases. Specificity constants (k(cat)/K(M)) of E. coli Nth protein were compared to those of its previously investigated functional homologues such as human and Schizosaccharomyces pombe Nth proteins and Saccharomyces cerevisiae Ntg1 and Ntg2 proteins. This comparison shows that significant differences exist with respect to substrate specificity and kinetic parameters despite extensive structural conservation among the Nth homologues.

Bacterial Proteins↗
PubMed2000

Induction of oxidative DNA damage in u937 cells by TNF or anti-Fas stimulation.

TNF and Fas signaling pathways are reported to induce mitochondrial damage associated with production of oxygen radicals. We examined whether such radical production elicited detectable nuclear DNA damage in U937 cells following treatment with TNF or with anti-Fas antibodies. Using GC-mass spectroscopy for analysing base oxidation, several oxidized species increased significantly following TNF treatment, whereas anti-Fas resulted in less detectable oxidative damage using this assay. Cytogenetic analysis showed that, in the presence of aphidicolin, which blocks several types of DNA repair, TNF induced extensive chromosomal damage. Aphidicolin also synergized with TNF and anti-Fas in inducing cell death which was prevented by reducing atmospheric oxygen or addition of n -acetyl cysteine, a scavenger of oxygen radicals. Thus, several lines of evidence point to the TNF and Fas pathways inducing extensive oxidative DNA damage and repair, and suggest potential roles for these pathways in mutagenesis and aging.

Adenosine↗
PubMed2000

Ni(II) specifically cleaves the C-terminal tail of the major variant of histone H2A and forms an oxidative damage-mediating complex with the cleaved-off octapeptide.

The acetyl-TESHHK-amide peptide, modeling a part of the C-terminal "tail" of histone H2A, was found previously by us to undergo at pH 7. 4 a Ni(II)-assisted hydrolysis of the E-S peptide bond with formation of a stronger Ni(II) complex with the SHHK-amide product [Bal, W., et al. (1998) Chem. Res. Toxicol. 11, 1014-1023]. To further characterize the hydrolysis and test the resulting Ni(II) complex for redox activity, bovine histone H2A and three peptides were investigated: acetyl-LLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK (H2A(34)), modeling the entire "C-tail" of H2A; SHHKAKGK (H2A(8)), modeling the cutoff product of hydrolysis; and acetyl-KTESHKAKGK (H2A(10)), modeling a putative Ni(II) binding site in a minor variant H2A.4 of human histone H2A. The Ni(II)-assisted hydrolysis of H2A and H2A(34) was found to proceed approximately 7-fold faster than that of the Ni(II)-acetyl-TESHHK-amide complex under comparable conditions. In both cases, the Ni(II) complex with H2A(8) was the smaller product of the hydrolysis, indicating a high site specificity of the reaction. Of three other metals tested with H2A(34), only Cu(II) cleaved the E-S bond, although much less efficiently than Ni(II); Co(II) and Zn(II) had no effect whatsoever. The H2A(10) peptide appeared to be fully resistant to hydrolytic cleavage and did not exhibit any redox activity versus H(2)O(2) in the presence of Ni(II) at pH 7.4. Likewise, redox-inactive was the Ni(II)-H2A(34) complex. In contrast, the Ni(II)-H2A(8) complex promoted oxidative damage of pUC19 DNA by H(2)O(2), evidenced by a significant increase in the number of single strand breaks and nucleobase modifications typical for a hydroxyl radical-like species attack on DNA. Interestingly, instead of 8-oxopurines, the corresponding formamidopyrimidines were the major products of the damage. The difference in redox activity between the Ni(II)-H2A(34) and Ni(II)-H2A(8) complexes is most likely associated with their different geometries: octahedral and square planar, respectively. Incubation of the Ni(II)-H2A(8) complex with H(2)O(2) also resulted in degradation of the peptide ligand, especially at its Ser and His residues. Thus, binding of Ni(II) to the ESHHK motif of the histone H2A C-tail is damaging to the histone C-terminal tail and to histone-associated DNA. The results support a dual mechanism of Ni(II)-induced carcinogenesis, including both genotoxic and epigenetic effects.

Amino Acid Sequence↗
PubMed1999

Excision of oxidatively damaged DNA bases by the human alpha-hOgg1 protein and the polymorphic alpha-hOgg1(Ser326Cys) protein which is frequently found in human populations.

We have investigated the substrate specificity of the major nuclear form of the human Ogg1 protein, referred as alpha-hOgg1, for excision of damaged bases from DNA exposed to gamma-irradiation. Excision products were identified and quantified using gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The GST-alpha-hOgg1 protein used in this study is a fusion of alpha-hOgg1 to the C-terminus of the GST protein. The results show that GST-alpha-hOgg1 protein excises 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA exposed to gamma-irradiation in a solution saturated with N(2)O or air. Fourteen other lesions, including oxidised purines and pyrimidines, were not excised from these substrates. Catalytic constants were measured for the excision of 8-OH-Gua and FapyGua from DNA gamma-irradiated under N(2)O. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 4.47 x 10(-5)and 8.97 x 10(-5)(min(-1)nM(-1)), respectively. The substrate specificity and the catalytic parameters of the wild-type GST-alpha-hOgg1 protein were compared to that of a polymorphic form of alpha-hOgg1 harbouring a Ser-->Cys mutation at codon 326. In the Japanese population, 47.6% of individuals possess both alleles coding for the wild-type alpha-hOgg1-Ser(326)and mutant alpha-hOgg1-Cys(326)proteins. The GST-alpha-hOgg1-Cys(326)protein was purified and its substrate specificity was determined by GC/IDMS analysis. The results show that the GST-alpha-hOgg1-Cys(326)protein efficiently excises 8-OH-Gua and FapyGua from gamma-irradiated DNA. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 2. 82 x 10(-5)and 4.43 x 10(-5)(min(-1)nM(-1)), respectively. Furthermore, we compared the capacity of these two forms of alpha-hOgg1 to act on substrates containing 2,6-diamino-4-hydroxy-5- N -methylformamidopyrimidine (Me-FapyGua). The k (cat)/ K (m)values for excision of Me-FapyGua were 278 x 10(-5)and 319 x 10(-5)(min(-1)nM(-1)), respectively. Cleavage of 34mer oligodeoxyribonucleotides containing 8-OH-Gua, 8-hydroxyadenine or an apurinic/apyrimidinic site paired with a cytosine was also investigated. The results show that both GST-alpha-hOgg1-Ser(326)and GST-alpha-hOgg1-Cys(326)catalyse the various cleavage reactions at very similar rates. Furthermore, both proteins efficiently complement the mutator phenotype of the fpg mutY mutant of Escherichia coli.

Amino Acid Substitution↗
PubMed1999

Saccharomyces cerevisiae Ntg1p and Ntg2p: broad specificity N-glycosylases for the repair of oxidative DNA damage in the nucleus and mitochondria.

Saccharomyces cerevisiae possesses two functional homologues (Ntg1p and Ntg2p) of the Escherichia coli endonuclease III protein, a DNA base excision repair N-glycosylase with a broad substrate specificity directed primarily against oxidatively damaged pyrimidines. The substrate specificities of Ntg1p and Ntg2p are similar but not identical, and differences in their amino acid sequences as well as inducibility by DNA damaging agents suggest that the two proteins may have different biological roles and subcellular locations. Experiments performed on oligonucleotides containing a variety of oxidative base damages indicated that dihydrothymine, urea, and uracil glycol are substrates for Ntg1p and Ntg2p, although dihydrothymine was a poor substrate for Ntg2p. Vectors encoding Ntg1p-green fluorescent protein (GFP) and Ntg2p-GFP fusions under the control of their respective endogenous promoters were utilized to observe the subcellular targeting of Ntg1p and Ntg2p in S. cerevisiae. Fluorescence microscopy of pNTG1-GFP and pNTG2-GFP transformants revealed that Ntg1p localizes primarily to the mitochondria with some nuclear localization, whereas Ntg2p localizes exclusively to the nucleus. In addition, the subcellular location of Ntg1p and Ntg2p confers differential sensitivities to the alkylating agent MMS. These results expand the known substrate specificities of Ntg1p and Ntg2p, indicating that their base damage recognition ranges show distinct differences and that these proteins mediate different roles in the repair of DNA base damage in the nucleus and mitochondria of yeast.

Base Sequence↗
PubMed1999

Repair of oxidative DNA base lesions induced by fluorescent light is defective in xeroderma pigmentosum group A cells.

Fluorescent light (FL) has been shown to generate free radicals within cells, however, the specific chemical nature of DNA damage induced by FL has not previously been determined. Using gas chromatography/isotope dilution mass spectrometry, we have detected induction of the oxidative DNA lesions 5-hydroxycytosine (5-OH-Cyt), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde) in cultured cells irradiated with FL. We followed the repair of these lesions in normal and xeroderma pigmentosum group A (XP-A) cells. 5-OH-Cyt and FapyGua were repaired efficiently in normal cells within 6 h following FL exposure. XP-A cells were unable to repair these oxidative DNA base lesions. Additionally, to compare the repair of oxidative lesions induced by various sources, in vitro repair studies were performed using plasmid DNA damaged by FL, gamma-irradiation or OsO(4)treatment. Whole cell extracts from normal cells repaired damaged substrates efficiently, whereas there was little repair in XP-A extracts. Our data demon-strate defective repair of oxidative DNA base lesions in XP-A cells in vivo and in vitro.

Cells, Cultured↗