Mutation Research/DNA Repair最新文献

{"title":"Backbone dynamics of DNA containing 8-oxoguanine: importance for substrate recognition by base excision repair glycosylases","authors":"M.L. Dodson,&nbsp;R.Stephen Lloyd","doi":"10.1016/S0921-8777(01)00109-4","DOIUrl":"10.1016/S0921-8777(01)00109-4","url":null,"abstract":"<div><p><span><span>Except for the functional groups sited within the major or minor grooves, the bases of B-DNA are quite protected from the external environment. Enzymes that modify the bases often “flip out” the target into an extrahelical position before the chemistry step is carried out. Examples of this mechanism are the base excision repair<span> glycosylases and the restriction enzyme methylases. The question arises about the mechanism of substrate recognition for these enzymes and how closely it is linked to the base flipping step. Molecular dynamics simulations (AMBER, PME electrostatics) of fully solvated, cation neutralized, </span></span>DNA sequences containing 8-oxoguanine (8OG) and of appropriate normal (control) DNAs have been carried out. The dynamics trajectories were analyzed to identify those properties of the DNA structure in the vicinity of the altered base, or its dynamics, that could contribute to molecular discrimination between substrate and non-substrate DNA sites. The results predict that the FPG enzyme should flip out the </span>cytosine base paired with the scissile 8OG, not the target base itself.</p></div>","PeriodicalId":100935,"journal":{"name":"Mutation Research/DNA Repair","volume":"487 3","pages":"Pages 93-108"},"PeriodicalIF":0.0,"publicationDate":"2001-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0921-8777(01)00109-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"56180289","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Escherichia coli recA mutant","authors":"Kazuhiro Kiyosawa ,&nbsp;Masashi Tanaka ,&nbsp;Tsukasa Matsunaga ,&nbsp;Osamu Nikaido ,&nbsp;Kazuo Yamamoto","doi":"10.1016/S0921-8777(01)00114-8","DOIUrl":"10.1016/S0921-8777(01)00114-8","url":null,"abstract":"<div><p>When a <em>recA</em> strain of <em>Escherichia coli</em> was transformed with the multicopy plasmid pSF11 carrying the <em>uvrA</em> gene of <em>E. coli</em>, its extreme ultraviolet (UV) sensitivity was decreased. The sensitivity of the <em>lexA1</em> (Ind⁻) strain to UV was also decreased by pSF11. The <em>recA</em> cells expressing <span><em>Neurospora</em><em> crassa</em></span> UV damage endonuclease (UVDE), encoding UV-endonuclease, show UV resistance. On the other hand, only partial amelioration of UV sensitivity of the <em>recA</em> strain was observed in the presence of the plasmid pNP10 carrying the <em>uvrB</em> gene. Host cell reactivation of UV-irradiated λ phage in <em>recA</em><span> cells with pSF11 was as efficient as that in wild-type cells. Using an antibody to detect cyclobutane pyrimidine dimers, we found that UV-irradiated </span><em>recA</em> cells removed dimers from their DNA more rapidly if they carried pSF11 than if they carried a vacant control plasmid. Using anti-UvrA antibody, we observed that the expression level of UvrA protein was about 20-fold higher in the <em>recA</em> strain with pSF11 than in the <em>recA</em> strain without pSF11. Our results were consistent with the idea that constitutive level of UvrA protein in the <em>recA</em><span> cells results in constitutive levels of active UvrABC nuclease<span> which is not enough to operate full nucleotide excision repair (NER), thus leading to extreme UV sensitivity.</span></span></p></div>","PeriodicalId":100935,"journal":{"name":"Mutation Research/DNA Repair","volume":"487 3","pages":"Pages 149-156"},"PeriodicalIF":0.0,"publicationDate":"2001-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0921-8777(01)00114-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"56180344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Excision of uracil from DNA by the hyperthermophilic Afung protein is dependent on the opposite base and stimulated by heat-induced transition to a more open structure","authors":"Ingeborg Knævelsrud ,&nbsp;Peter Ruoff ,&nbsp;Hilde Ånensen ,&nbsp;Arne Klungland ,&nbsp;Svein Bjelland ,&nbsp;Nils-Kåre Birkeland","doi":"10.1016/S0921-8777(01)00115-X","DOIUrl":"10.1016/S0921-8777(01)00115-X","url":null,"abstract":"<div><p><span><span>Hydrolytic deamination of DNA-cytosines into </span>uracils<span> is a major source of spontaneously induced mutations<span><span>, and at elevated temperatures the rate of cytosine deamination is increased. Uracil lesions are repaired by the base excision repair pathway, which is initiated by a specific uracil DNA glycosylase enzyme (UDG). The </span>hyperthermophilic archaeon </span></span></span><span><em>Archaeoglobus fulgidus</em></span> contains a recently characterized novel type of UDG (Afung), and in this paper we describe the over-expression of the <em>afung</em> gene and characterization of the encoded protein. Fluorescence and activity measurements following incubation at different temperatures may suggest the following model describing structure-activity relationships: At temperatures from 20 to 50<!--> <span>°C Afung exists as a compact protein exhibiting low enzyme activity, whereas at temperatures above 50</span> <!-->°C, the Afung conformation opens up, which is associated with the acquisition of high enzyme activity. The enzyme exhibits opposite base-dependent excision of uracil in the following order: U&gt;U:T&gt;U:C⪢U:G⪢U:A. Afung is product-inhibited by uracil and shows a pronounced inhibition by <em>p</em>-hydroxymercuribenzoate, indicating a cysteine residue essential for enzyme function. The Afung protein was estimated to be present in <em>A. fulgidus</em> at a concentration of ∼1000<!--> <span>molecules per cell. Kinetic parameters determined for Afung suggest a significantly lower level of enzymatic uracil release in </span><em>A. fulgidus</em> as compared to the mesophilic <em>Escherichia coli</em>.</p></div>","PeriodicalId":100935,"journal":{"name":"Mutation Research/DNA Repair","volume":"487 3","pages":"Pages 173-190"},"PeriodicalIF":0.0,"publicationDate":"2001-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0921-8777(01)00115-X","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"56180355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transcriptional induction of repair genes during slowing of replication in irradiated Saccharomyces cerevisiae","authors":"G. Mercier ,&nbsp;Y. Denis ,&nbsp;P. Marc ,&nbsp;L. Picard ,&nbsp;M. Dutreix","doi":"10.1016/S0921-8777(01)00116-1","DOIUrl":"10.1016/S0921-8777(01)00116-1","url":null,"abstract":"<div><p><span>We investigated the inhibition of cell-cycle progression and replication and the induction of the transcriptional response in diploid budding yeast populations exposed to two different doses of γ-rays resulting in 15 and 85% survival respectively. We studied the kinetics of the cellular response to ionizing treatment during the period required for all of the surviving cells to achieve at least one cell division. The length of these periods increased with the dose. Irradiated populations arrested as large-budded cells containing partially replicated chromosomes. The extent of the S-phase was proportional to the amount of damage and lasted 3 or 7</span> <!-->h depending on the irradiation dose. In parallel to the division study, we carried out a kinetic analysis of the expression of 126 selected genes by use of dedicated microarrays. About 26 genes were induced by irradiation and displayed various pattern of expression. Interestingly, 10 repair genes (<span><em>RAD51</em></span>, <em>RAD54</em>, <em>CDC8</em>, <span><em>MSH2</em></span>, <em>RFA2</em>, <em>RFA3</em>, <em>UBC5</em>, <em>SRS2</em>, <em>SPO12</em> and <span><em>TOP1</em></span><span>), involved in recombination and DNA synthesis, display similar regulation of expression in the two irradiated populations. Their pattern of expression were confirmed by Northern analysis. At the two doses, the expression of this group of genes closely followed the extended replication period, and their expression resumed when replication restarted. These results suggest that the damage-induced response and DNA synthesis are closely regulated during repair. The analysis of the promoter regions indicates a high occurrence of the three MCB, HAP and UASH regulatory boxes in the promoters of this group of genes. The association of the three boxes could confer an irradiation-replication specific regulation.</span></p></div>","PeriodicalId":100935,"journal":{"name":"Mutation Research/DNA Repair","volume":"487 3","pages":"Pages 157-172"},"PeriodicalIF":0.0,"publicationDate":"2001-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0921-8777(01)00116-1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"56180365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spontaneous and ultraviolet light-induced direct repeat recombination in mammalian cells frequently results in repeat deletion","authors":"Colin A. Bill,&nbsp;Jac A. Nickoloff","doi":"10.1016/S0921-8777(01)00101-X","DOIUrl":"10.1016/S0921-8777(01)00101-X","url":null,"abstract":"<div><p><span>Recombination is enhanced by transcription and by DNA damage caused by ultraviolet light (UV). Recombination between direct repeats can occur by gene conversion without an associated crossover, which maintains the gross repeat structure. There are several possible mechanisms that delete one repeat and the intervening sequences (gene conversion associated with a crossover, unequal sister chromatid exchange<span>, and single-strand annealing). We examined transcription-enhanced spontaneous recombination, and UV-induced recombination between neomycin (</span></span><em>neo</em>) direct repeats. One <em>neo</em> gene was driven by the inducible MMTV promoter. Multiple (silent) markers in the second <em>neo</em> gene were used to map conversion tracts. These markers are thought to inhibit spontaneous recombination, and our data suggest that this inhibition is partially overcome by high level transcription. Recombination was stimulated by transcription and by UV doses of 6–12<!--> <!-->J/m², but not by 18<!--> <!-->J/m². About 70% of spontaneous and UV-induced products were deletions. In contrast, only 3% of DSB-induced products were deletions. We propose that these product spectra differ because spontaneous and UV-induced recombination is replication-dependent, whereas DSB-induced recombination is replication-independent.</p></div>","PeriodicalId":100935,"journal":{"name":"Mutation Research/DNA Repair","volume":"487 1","pages":"Pages 41-50"},"PeriodicalIF":0.0,"publicationDate":"2001-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0921-8777(01)00101-X","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"56180170","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Accumulation of oxidative DNA damage, 8-oxo-2′-deoxyguanosine, and change of repair systems during in vitro cellular aging of cultured human skin fibroblasts","authors":"Takao Kaneko ,&nbsp;Shoichi Tahara ,&nbsp;Takahiko Taguchi ,&nbsp;Hiroshi Kondo","doi":"10.1016/S0921-8777(01)00100-8","DOIUrl":"10.1016/S0921-8777(01)00100-8","url":null,"abstract":"<div><p><span>Effects of in vitro cellular aging on the content of 8-oxo-2′-deoxyguanosine, a typical oxidation product of DNA bases, were examined in cultured human skin fibroblasts. The 8-oxo-2′-deoxyguanosine content in the DNA of TIG-3S cells established from skin tissues of a fetal donor increased immediately before the cessation of proliferation. TIG-114 and TIG-104 cells established from skin tissues of adult and aged donors, respectively, showed similar changes in 8-oxo-2′-deoxyguanosine content during in vitro cellular aging. The accumulation of 8-oxo-2′-deoxyguanosine in late-passage cells was dependent on the number of cell divisions, and not on the cultivation time. Increases in the activities of superoxide dismutase and </span>glutathione<span> peroxidase were observed prior to the increase in 8-oxo-2′-deoxyguanosine content, while the catalase<span> activity decreased gradually during in vitro cellular aging at late-passage. Furthermore, the activities of 8-oxo-2′-deoxyguanosine endonuclease and DNA polymerases decreased with the progression of proliferation. These results indicate that defense systems against oxidative stress in late-passage cells remain sufficiently active before the cessation of cell division, but that repair systems against oxidative damage decay at late-passage. Oxidative stress beyond the antioxidant capacity and/or repair activity seems to result in an accumulation of 8-oxo-2′-deoxyguanosine in late-passage cells.</span></span></p></div>","PeriodicalId":100935,"journal":{"name":"Mutation Research/DNA Repair","volume":"487 1","pages":"Pages 19-30"},"PeriodicalIF":0.0,"publicationDate":"2001-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0921-8777(01)00100-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"56180152","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"SUVi and BACH1: a new subfamily of mammalian helicases?","authors":"Paola Menichini ,&nbsp;Michal Linial","doi":"10.1016/S0921-8777(01)00104-5","DOIUrl":"10.1016/S0921-8777(01)00104-5","url":null,"abstract":"<div><p>The RecQ family of DNA helicases have been shown to be important for the maintenance of genomic integrity in prokaryotes<span> and eukaryotes. Members of this family are genes responsible for cancer predisposition disorders like Bloom’s syndrome, Werner’s syndrome and Rothmund-Thomson syndrome. Here, we show the sequence homologies<span> between two recently identified mammalian helicases, namely SUVi and BACH1. These two genes also share strong homologies with other members of the RecQ family. On the basis of published data and sequence analysis we suggest that SUVi/BACH1 may represent a novel subfamily of mammalian helicases, functioning in the processing of lesions induced by different genotoxic agents.</span></span></p></div>","PeriodicalId":100935,"journal":{"name":"Mutation Research/DNA Repair","volume":"487 1","pages":"Pages 67-71"},"PeriodicalIF":0.0,"publicationDate":"2001-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0921-8777(01)00104-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"56180228","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Repair of O6-methylguanine is not affected by thymine base pairing and the presence of MMR proteins","authors":"Jochen Lips,&nbsp;Bernd Kaina","doi":"10.1016/S0921-8777(01)00105-7","DOIUrl":"10.1016/S0921-8777(01)00105-7","url":null,"abstract":"<div><p><span>Methylation at the </span><em>O</em>⁶<span>-position of guanine (</span><em>O</em>⁶-MeG) by alkylating agents is efficiently removed by <em>O</em>⁶-methylguanine–DNA methyltransferase (MGMT), preventing from cytotoxic, mutagenic, clastogenic and carcinogenic effects of <em>O</em>⁶-MeG-inducing agents. If <em>O</em>⁶<span><span>-MeG is not removed from DNA prior to replication, thymine will be incorporated instead of </span>cytosine opposite the </span><em>O</em>⁶<span><span>-MeG lesion. This mismatch is recognized and processed by mismatch repair (MMR) proteins which are known to be involved in triggering the cytotoxic and genotoxic response of cells upon methylation. In this work we addressed three open questions. (1) Is </span>MGMT able to repair </span><em>O</em>⁶-MeG mispaired with thymine (<em>O</em>⁶-MeG/T)? (2) Do MMR proteins interfere with the repair of <em>O</em>⁶-MeG/T by MGMT? (3) Does MGMT show a protective effect if it is expressed after replication of DNA containing <em>O</em>⁶<span><span>-MeG? Using an in vitro assay we show that </span>oligonucleotides containing </span><em>O</em>⁶-MeG/T mismatches are as efficient as oligonucleotides containing <em>O</em>⁶-MeG/C in competing for MGMT repair activity, indicating that <em>O</em>⁶-MeG mispaired with thymine is still subject to repair by MGMT. The addition of MMR proteins from nuclear extracts, or of recombinant MutSα, to the in vitro repair assay did not affect the repair of <em>O</em>⁶-MeG/T lesions by MGMT. This indicates that the presence of MutSα still allows access of MGMT to <em>O</em>⁶-MeG/T lesions. To elucidate the protective effect of MGMT in the first and second replication cycle after <em>N</em>-methyl-<em>N</em>′-nitro-<em>N</em>-nitrosoguanidine (MNNG) treatment, MGMT transfected CHO cells were synchronized and MGMT was inactivated by pulse-treatment with <em>O</em>⁶-benzylguanine (<em>O</em>⁶-BG). Thereafter, the recovered cells were treated with MNNG and subjected to clonogenic survival assays. Cells which expressed MGMT in the first and second cell cycle were more resistant than cells which expressed MGMT only in the second (post-treatment) cell cycle. Cells which did not express MGMT in both cell cycles were most sensitive. This indicates that repair of <em>O</em>⁶<span>-MeG can occur both in the first and second cell cycle after alkylation protecting cells from the killing effect of the lesion.</span></p></div>","PeriodicalId":100935,"journal":{"name":"Mutation Research/DNA Repair","volume":"487 1","pages":"Pages 59-66"},"PeriodicalIF":0.0,"publicationDate":"2001-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0921-8777(01)00105-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"56180238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phenotype of FAECB (Facility for Automated Experiments in Cell Biology) Chinese hamster ovary mutants with minimal UV-sensitivity","authors":"David B Busch ,&nbsp;Deborah White Ziffer ,&nbsp;Donna Coleman ,&nbsp;Lisa Wills ,&nbsp;H Greg McDonough ,&nbsp;Nigel J Jones","doi":"10.1016/S0921-8777(01)00099-4","DOIUrl":"10.1016/S0921-8777(01)00099-4","url":null,"abstract":"<div><p><span>The Facility for Automated Experiments in Cell Biology (FAECB) collection of over 200 lines of ultraviolet (UV)-sensitive mutant Chinese hamster ovary (CHO) cells has previously been studied for complementation group assignment (CG), with representatives of rodent UV CGs 1–6 (</span><em>ERCC1–6)</em> and the new rodent <em>XRCC9/FANCG</em> group identified. Ten mutants from the collection, including a further six derived from wildtype AA8, three UV-sensitive double-mutants of CHO <span><em>ERCC1</em></span> cell line UV4, and a UV-sensitive mutant of CHO <span><em>XRCC1</em></span><span> cell line EM9, had not been assigned or characterized in these previous studies. These 10 mutants include 8 with approximately 1.5-fold the UV-sensitivity of the parental line (AA8, EM9, or UV4), and 2 with about 2-fold the UV-sensitivity of AA8. The present study reports the partial characterization of these 10 mutants in terms of sensitivity to UV (with and without caffeine), ionizing radiation, mitomycin C (MMC) and ethyl methanesulfonate<span> (EMS); proficiency in DNA repair (unscheduled DNA synthesis (UDS)); and UV-mutability. The phenotypes of the 10 cell lines were heterogeneous, a number showed reduced UDS or UV-sensitization by caffeine, whilst others showed marked sensitivity to EMS or MMC, and they may have mutations in different genes involved in nucleotide excision repair<span><span>, post-replicational repair, base excision repair or recombinational repair. Previous mutants isolated as part of the FAECB collection have proved to be extremely important in characterizing mammalian DNA repair processes and </span>cloning human repair genes and these current mutants, whilst not as hypersensitive to UV, may still have the potential to make further contributions.</span></span></span></p></div>","PeriodicalId":100935,"journal":{"name":"Mutation Research/DNA Repair","volume":"487 1","pages":"Pages 31-39"},"PeriodicalIF":0.0,"publicationDate":"2001-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0921-8777(01)00099-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"56180143","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Overexpression of Ogt reduces MNU and ENU induced transition, but not transversion, mutations in E. coli","authors":"Karen Beenken ,&nbsp;Zhehong Cai,&nbsp;Douglas Fix","doi":"10.1016/S0921-8777(01)00102-1","DOIUrl":"10.1016/S0921-8777(01)00102-1","url":null,"abstract":"<div><p>Studies of alkylation-induced mutations in <em>Escherichia coli</em> FX-11 revealed that both <em>N</em>-ethyl-<em>N</em>-nitrosourea (ENU) and <em>N</em>-methyl-<em>N</em><span>-nitrosourea (MNU) produced tRNA suppressor mutations (G:C to A:T) but only ENU produced a significant number of backmutations (A:T to G:C, A:T to T:A and A:T to C:G). Further, the ENU-induced transversions<span><span><span> were absent in a UmuC-defective strain. This suggested that transition mutations could result from alkylation of </span>guanine or </span>thymine at the </span></span><em>O</em>⁶- and <em>O</em>⁴-positions, respectively, but that transversions might result from alkylation of thymine at the <em>O</em>²-position. To test this idea, the gene encoding <em>O</em>⁶-alkylguanine-DNA methyltransferase (<em>ogt</em>) was recombined into a plasmid to overexpress the cellular levels of this enzyme. Ogt protein can de-alkylate <em>O</em>⁶-alkylguanine and <em>O</em>⁴-alkylthymine, but not <em>O</em>²-alkylthymine. Cells harboring the plasmid (or a control plasmid lacking the <em>ogt</em> gene) were exposed to different concentrations of MNU or ENU and the resulting mutations were analyzed. With either MNU or ENU, the frequency of GlnV^o suppressors was reduced about 70-fold in the Ogt-overexpressing cells, suggesting that Ogt eliminated <em>O</em>⁶-alkylguanine. Similarly, GlnU^o<span> suppressor frequencies were substantially reduced. In contrast, the reduction in frequency for the backmutations was slight, only about 2.5-fold with MNU and less than two-fold for ENU. However, DNA sequence analysis of the backmutations showed that only A:T to G:C transitions were affected by overexpression of Ogt, suggesting repair of </span><em>O</em>⁴-alkylthymine. The frequency of transversions, in comparison, was essentially unaltered. These results implicate <em>O</em>²<span>-alkylthymine as a likely candidate for transversion mutagenesis induced by ENU.</span></p></div>","PeriodicalId":100935,"journal":{"name":"Mutation Research/DNA Repair","volume":"487 1","pages":"Pages 51-58"},"PeriodicalIF":0.0,"publicationDate":"2001-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0921-8777(01)00102-1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"56180215","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}