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J M Essigmann

Publications and source records attributed to J M Essigmann.

At least 73 records · Page 4Linked to original sources
PubMed1990

Comparative efficiency of forming m4T.G versus m4T.A base pairs at a unique site by use of Escherichia coli DNA polymerase I (Klenow fragment) and Drosophila melanogaster polymerase alpha-primase complex.

Synthesis of a 25-mer oligonucleotide template containing O4-methylthymine (m4T) at a unique site is reported. The sequence used is analogous to that studied previously to determine the mutation frequency of O6-methylguanine in vitro and in vivo. The templates containing m4T or unmodified T were used in a primer-extension gel assay to determine kinetic parameters for incorporation by DNA polymerases of dGTP and dATP opposite either m4T or T. Both Escherichia coli DNA polymerase I (Klenow fragment, Kf) and Drosophila melanogaster polymerase alpha-primase complex (pol alpha) were used. On the basis of the Vmax/Km ratios, the pairing of m4T.G was preferred over that of both m4T.A and T.G by more than 10-fold. The two polymerases gave almost identical values for the frequency of formation of all pairs investigated including m4T.G pairs, suggesting that the 3'----5' exonuclease activity of the Klenow fragment does not efficiently edit such pairs. Extension beyond m4T.G was demonstrated with both Klenow and pol alpha. In similar kinetic experiments, bacteriophage T4 DNA polymerase, which has a very high 3'----5' exonuclease activity, allows stable incorporation of G opposite m4T in contrast to G opposite T. This kinetic approach allows quantitation of the mutagenic potential in the absence of alkylation repair and additionally provides qualitative data on mutagenesis that are in accord with our previous in vivo studies showing that replication of m4T causes T----C transitions.

Animals↗
PubMed1989

Genetic effects of thymine glycol: site-specific mutagenesis and molecular modeling studies.

The mutational specificity and genetic requirements for mutagenesis by 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), one of the principal DNA lesions induced by oxidation and ionizing radiation, has been investigated in Escherichia coli. Thymine glycol was positioned at a unique site in the single-stranded genome of a bacteriophage M13mp19 derivative. Replication of the genome in E. coli yielded targeted mutations at a frequency of 0.3%; the mutations were exclusively T----C. Mutagenesis was independent of SOS and nth (nth encodes endonuclease III, a thymine glycol repair enzyme). The adduct was not detectably mutagenic in duplex DNA. A chemical rationalization for the mutation observed for thymine glycol was developed by applying molecular modeling and molecular mechanical calculations to the same DNA sequence studied in vivo. Modeling suggested that the 5R,6S isomer of cis-thymine glycol, when not base paired, was displaced laterally by approximately 0.5 A toward the major groove in comparison to the position that thymine would otherwise occupy. This perturbation of DNA structure should increase the likelihood of a guanine.thymine glycol wobble base pair during replication, which would explain the mutational specificity of the base observed in the genetic experiments.

Base Sequence↗
PubMed1989

Effect of 3' flanking neighbors on kinetics of pairing of dCTP or dTTP opposite O6-methylguanine in a defined primed oligonucleotide when Escherichia coli DNA polymerase I is used.

O6-Methylguanine (m6G) was incorporated site-specifically into two 25-base oligonucleotides differing only in the nucleotide on the 3' side of the modified base. Templates were primed with oligonucleotides terminating one or two bases prior to the site at which incorporation kinetics were to be investigated. Escherichia coli DNA polymerase I (Klenow fragment) was used to determine the apparent Km and relative Vmax of incorporation of either dCTP or dTTP opposite m6G or G. These data were used to calculate the relative frequency of incorporation opposite the m6G or the unmodified G. When the sequence was 3'-Cm6G-5', there was a 6- to 7-fold preference for formation of a m6G.T pair compared with m6G.C. The m6G.T frequency, based on Vmax/Km, was at least 50-fold greater than that of a G.T pair at the same site. Changing the sequence to 3'-Tm6G-5' had a marked effect on both Km and Vmax of pairs containing m6G and on the incorporation frequency of T opposite m6G, which was then only slightly favored over m6G.C. When replication was started directly opposite m6G, the kinetics appeared unaffected. These data indicate that the frequency of incorporation of C or T opposite m6G in a DNA template is dependent on the flanking neighbors and that a change of even a single base at the 3' position can have a major effect on mutagenic efficiency. Replication using Drosophila Pol alpha gave the same values for relative frequencies. Pairing of either C or T with m6G on the primer terminus did not significantly inhibit extension of the next normal base pair, in contrast to terminal mismatches of unmodified bases. It is concluded that, in the absence of repair, m6G can exhibit widely differing mutation frequencies which, in these experiments, can be as high as 85% of the replicated base. This variation in frequency of changed pairing could contribute to the occurrence of mutational "'hot spots" after replication of damaged DNA.

Base Composition↗
PubMed1989

Isolation of cDNAs encoding a human protein that binds selectively to DNA modified by the anticancer drug cis-diamminedichloroplatinum(II)

DNA modified by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) was used to identify a factor in mammalian cells that binds to cis-DDP-damaged DNA and hence may play a role in repair. This factor selectively recognizes double-stranded DNA fragments modified by cis-DDP or [Pt(en)Cl2] (en, ethylenediamine). Little or no binding occurs to unmodified double-stranded DNA or to DNA modified with the clinically ineffective compounds trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine). Low levels of binding to single-stranded DNA modified by cis-DDP are observed. The apparent molecular mass of the factor in a variety of mammalian cells is approximately 100 kDa, as determined by modified Western blotting. Two recombinant phage have been isolated from a human B-cell lambda gt11 library by using a cis-DDP-modified DNA restriction fragment as a probe. The two clones have insert sizes of 1.88 and 1.44 kilobases and are aligned at their 5' ends. The polypeptides encoded by the recombinant phage exhibit DNA binding properties similar to those of the cellular factor identified in crude extracts prepared from mammalian cells. Northern analysis with one of the clones revealed an mRNA of 2.8 kilobases that is conserved in humans and rodents. The methods used here should be applicable in studies of other damage-specific DNA binding proteins.

Bacteriophage lambda↗
PubMed1989

Site-specific mutagenesis by O6-alkylguanines located in the chromosomes of mammalian cells: influence of the mammalian O6-alkylguanine-DNA alkyltransferase.

A plasmid was constructed in which a single guanine residue was replaced with either O6-methylguanine or O6-ethylguanine, two of the DNA adducts formed by carcinogenic alkylating agents. The vectors were introduced in parallel into a pair of Chinese hamster ovary cells, in which one member of the pair was deficient in the repair enzyme O6-alkylguanine-DNA alkyltransferase (mex-) and the other was proficient in this activity (mex+). The vectors integrated into and replicated within the respective host genomes. After intrachromosomal replication, the DNA sequence in the vicinity of the originally adducted site of each integrated vector was amplified from the host genome by using the polymerase chain reaction and was analyzed for mutations. High levels of mutation were observed from the O6-methylguanine- and O6-ethylguanine-containing vectors replicated in mex- cells (approximately 19% for O6-methylguanine and approximately 11% for O6-ethylguanine). DNA sequencing revealed the induced mutations to be almost exclusively G----A transitions. By contrast, little or no mutagenesis was detected when the adducted vectors were introduced into mex+ cells, indicating the significant role of the O6-alkylguanine-DNA alkyltransferase in the repair of O6-methylguanine and O6-ethylguanine in these mammalian cells.

Animals↗
PubMed1989

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.

DNA Repair↗
PubMed1989

Mutagenic specificity of alkylated and oxidized DNA bases as determined by site-specific mutagenesis.

This work demonstrates the use of the tools of site-specific mutagenesis to study the mutagenic activity of two DNA adducts, O6-methylguanine and cis-thymine glycol. The former adduct is one of the methylated bases formed by carcinogenic and mutagenic alkylating agents. It was built into the single-stranded genome of bacteriophage M13 and replicated in Escherichia coli (E. coli). The mutation frequency of O6-methylguanine was 0.4% in physiologically normal cells. In cells in which the repair systems for O6-methylguanine were compromised by challenge with an alkylating agent, the mutation frequency rose to approximately 20%. DNA sequencing revealed that O6-methylguanine induced exclusively G----A transitions, which was most consistent with it pairing with thymine during DNA synthesis. The mutagenic effects also were investigated of cis-thymine glycol isomers, which are major, stable products of ionizing radiation and oxidative damage to DNA. By techniques similar to those employed for the study of O6-methylguanine mutagenesis, a single thymine glycol was situated in an M13 phage genome. The genome was replicated in E. coli that were physiologically normal, induced for SOS functions, or deficient in the nth gene product and, in all cases, the mutagenic processing of thymine glycol in vivo yielded mutant progeny phage at a frequency of 0.3-0.4%. All mutations occurred at the site that originally contained thymine glycol, and all were demonstrated by DNA sequencing to have resulted from targeted T----C transitions. These data suggest that thymine glycol pairs with guanine during replication.

DNA Mutational Analysis↗
PubMed1988

Specificity of mutagenesis by 4-aminobiphenyl. A possible role for N-(deoxyadenosin-8-yl)-4-aminobiphenyl as a premutational lesion.

Mutagenesis by N-acetoxy-N-trifluoroacetyl-4-aminobiphenyl, a reactive form of the human bladder carcinogen 4-aminobiphenyl (ABP), was studied in Escherichia coli virus M13mp10. N-acetoxy-N-trifluoroacetyl-4-ABP-treated DNA containing 140 lesions/duplex genome, when introduced into excision repair-competent cells induced for SOS mutagenic processing, resulted in a 40-fold increase in mutation frequency over background in the lacZ alpha gene fragment. DNA sequence changes were determined for 20 independent mutants. G-C base pairs were the major targets for base pair substitution mutations, although significant mutagenic activity was also observed at certain A-T base pairs. Deletion and frameshift mutations also were found in this sample. The salient feature of this partial "mutational spectrum" was a hotspot that occurred at position 6357 (amino acid 30 of the beta-galactosidase fragment encoded by M13mp10); this A-T to T-A transversion appeared in 6 of the 20 mutants. The property of ABP to mutate A-T base pairs was consistent with the result that N-hydroxy-ABP reverted Salmonella typhimurium strain TA104, which is presumed to revert primarily due to mutations at these sites. The ability of the major carcinogen-DNA adduct formed by ABP in vivo and in vitro, N-(deoxyguanosin-8-yl)-4-aminobiphenyl, to cause base pair substitution mutations was also investigated. This adduct was positioned specifically in the minus strand at position 6270 in duplex M13mp10 DNA. In the presence of the mutagenesis-enhancing plasmid pGW16 and UV induction of SOS mutagenic processing, it was shown that fewer than 0.02% of the adducts resulted in transition or transversion mutations following transfection of DNA into excision-repair competent cells. Similar results were obtained in uvrA and uvrC backgrounds. Although the major adduct did not cause base substitution mutations under these experimental conditions, the contribution of this lesion to the entire spectrum of mutations in the lacZ alpha fragment seems likely.

Amino Acid Sequence↗
PubMed1988

Chemical and biological studies of the major DNA adduct of cis-diamminedichloroplatinum(II), cis-[Pt(NH3)2(d(GpG]], built into a specific site in a viral genome.

A duplex Escherichia coli bacteriophage M13 genome was constructed containing a single cis-[Pt(NH3)2(d(GpG]] intrastrand cross-link, the major DNA adduct of the anticancer drug cis-diamminedichloroplatinum(II). The duplex dodecamer d(AGAAGGCCTAGA).d(TCTAGGCCTTCT) was ligated into the HincII site of M13mp18 to produce an insertion mutant containing a unique StuI restriction enzyme cleavage site. A genome with a 12-base gap in the minus strand was created by hybridizing HincII-linearized M13mp18 duplex DNA with the single-stranded circular DNA of the 12-base insertion mutant. The dodecamer d(TCTAGGCCTTCT) was synthesized by the solid-phase phosphotriester method and platinated by reaction with cis-[Pt(NH3)2(H2O)2]2+ (yield 39%). Characterization by pH-dependent 1H NMR spectroscopy established that platinum binds to the N7 positions of the adjacent guanosines. The platinated oligonucleotide was phosphorylated in the presence of [gamma-32P]ATP with bacteriophage T4 polynucleotide kinase and incorporated into the 12-base gap of the heteroduplex, thus situating the adduct specifically within the StuI site in the minus strand of the genome. Approximately 80% of the gapped duplexes incorporated a dodecanucleotide in the ligation reaction. Of these, approximately half did so with the dodecanucleotide covalently joined to the genome at both 5' and 3' termini. The site of incorporation of the dodecamer was mapped to the expected 36-base region delimited by the recognition sites of XbaI and HindIII. The cis-[Pt(NH3)2(d(GpG]] cross-link completely inhibited StuI cleavage, which was fully restored following incubation of the platinated genome with cyanide to remove platinum as [Pt(CN)4]2-. Gradient denaturing gel electrophoresis of a 289-base-pair fragment encompassing the site of adduction revealed that the presence of the cis-[Pt(NH3)2(d(GpG]] cross-link induces localized weakening of the DNA double helix. In addition, double- and single-stranded genomes, in which the cis-[Pt(NH3)2(d(GpG]] cross-link resides specifically in the plus strand, were constructed. Comparative studies revealed no difference in survival between platinated and unmodified double-stranded genomes. In contrast, survival of the single-stranded platinated genome was only 10-12% that of the corresponding unmodified single-stranded genome, indicating that the solitary cis-[Pt(NH3)2(d(GpG]] cross-link is lethal to the single-stranded bacteriophage.

Binding Sites↗
PubMed1988

O6-alkyldeoxyguanosine detection by 32P-postlabeling and nucleotide chromatographic analysis.

The 32P-postlabeling procedure, developed originally by Randerath and coworkers, has been modified for the detection and analytical quantitation of O6-alkyl-2'-deoxyguanosine residues in DNA. Chromatographic techniques were developed to resolve individually the normal deoxyribonucleotide-3'-monophosphates and the O6-alkyldeoxyguanosine-3'-monophosphates by high-pressure liquid chromatography. Selective deoxyribonucleotide-3'-monophosphates (e.g., O6-alkyldeoxyguanosine-3'-monophosphates) were then converted to labeled deoxyribonucleotide-[5'-32P]monophosphates by 32P-postlabeling and nuclease P1 treatment and separated by two-dimensional thin layer chromatography. The O6-methyl- and O6-ethyl-2'-deoxyguanosine-3'-monophosphate nucleotides, and the respective 5'-monophosphates, were chemically synthesized for standardization of these quantitative procedures. The quantitation of O6-methl- and O6-ethyl-2'-deoxyguanosine was observed to be analytically accurate between one O6-alkyl-2'-deoxyguanosine residue per 10(4) and 10(7) 2'-deoxyguanosines. The limit of detection was less than one O6-alkyl-2'-deoxyguanosine in 10(7) 2'-deoxyguanosine residues in a sample size of 100 micrograms of DNA, i.e., approximately 10 pg of adduct. The quantitation of O6-methyl-2'-deoxyguanosine in the liver DNAs of rats treated with [14C-Me]N-nitrosodimethylamine compared well with values obtained by both 14C and high-pressure liquid chromatography coupled with fluorescence detection. Thus, these 32P-postlabeling and nucleotide chromatographic procedures should be useful in monitoring human exposure to methylating and ethylating carcinogens.

Alkylation↗
PubMed1987

Deoxyhexanucleotide containing a vinyl chloride induced DNA lesion, 1,N6-ethenoadenine: synthesis, physical characterization, and incorporation into a duplex bacteriophage M13 genome as part of an amber codon.

Organic synthesis and recombinant DNA techniques have been used to situate a single 1,N6-ethenoadenine (epsilon Ade) DNA adduct at an amber codon in the genome of an M13mp19 phage derivative. The deoxyhexanucleotide d[GCT(epsilon A)GC] was chemically synthesized by the phosphotriester method. Mild nonaqueous conditions were employed for deprotection because of the unstable nature of the epsilon Ade adduct in aqueous basic milieu. Physical studies involving fluorescence, circular dichroism, and 1H NMR indicated epsilon Ade to be very efficiently stacked in the hexamer, especially with the 5'-thymine. Melting profile and circular dichroism studies provided evidence of the loss of base-pairing capabilities attendant with formation of the etheno ring. The modified hexanucleotide was incorporated into a six-base gap formed in the genome of an M13mp19 insertion mutant; the latter was constructed by blunt-end ligation of d(GCTAGC) in the center of the unique SmaI site of M13mp19. Phage of the insertion mutant, M13mp19-NheI, produced light blue plaques on SupE strains because of the introduced amber codon. Formation of a hybrid between the single-strand DNA (plus strand) of M13mp19-NheI with SmaI-linearized M13mp19 replicative form produced a heteroduplex with a six-base gap in the minus strand. The modified hexamer [5'-32P]d-[GCT(epsilon A)GC], after 5'-phosphorylation, was ligated into this gap by using bacteriophage T4 DNA ligase to generate a singly adducted genome with epsilon Ade at minus strand position 6274. Introduction of the radiolabel provided a useful marker for characterization of the singly adducted genome, and indeed the label appeared in the anticipated fragments when digested by several restriction endonucleases. Evidence that ligation occurred on both 5' and 3' sides of the oligonucleotide also was obtained. The adduct was introduced into a unique NheI site, and it was observed that this restriction endonuclease was able to cleave the adducted genome, albeit at a lower rate compared to unmodified DNA. The M13mp19-NheI genome containing epsilon Ade will be used as a probe for studying mutagenesis and repair of this DNA adduct in Escherichia coli.

Adenosine↗
PubMed1987

A probe for the mutagenic activity of the carcinogen 4-aminobiphenyl: synthesis and characterization of an M13mp10 genome containing the major carcinogen-DNA adduct at a unique site.

The duplex genome of Escherichia coli virus M13mp10 was modified at a unique site to contain N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG8-ABP), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG8-ABP residue was synthesized by reacting 5'-d(TpGpCpA)-3' with N-acetoxy-N-(trifluoracetyl)-4-aminobiphenyl, followed by high-performance liquid chromatography purification of the principal reaction product 5'-d(TpG8-ABPpCpA)-3' (yield 15-30%). Characterization by fast atom bombardment mass spectrometry confirmed the structure as an intact 4-aminobiphenyl-modified tetranucleotide, while 1H nuclear magnetic resonance spectroscopy established the site of substitution and the existence of ring stacking between the carcinogen residue and DNA bases. Both 5'-d(TpG8-ABPpCpA)-3' and 5'-d(TpGpCpA)-3' were 5'-phosphorylated by use of bacteriophage T4 polynucleotide kinase and were incorporated into a four-base gap uniquely positioned in the center of the recognition site for the restriction endonuclease PstI, in an otherwise duplex genome of M13mp10. In the case of the adducted tetranucleotide, dG8-ABP was located in the minus strand at genome position 6270. Experiments in which the tetranucleotides were 5' end labeled with [32P]phosphate revealed the following: the adducted oligomer, when incubated in a 1000-fold molar excess in the presence of T4 DNA ligase and ATP, was found to be incorporated into the gapped DNA molecules with an efficiency of approximately 30%, as compared to the unadducted d(pTpGpCpA), which was incorporated with 60% ligation efficiency; radioactivity from the 5' end of each tetranucleotide was physically mapped to a restriction fragment that contained the PstI site and represented 0.2% of the genome; the presence of the lesion within the PstI recognition site inhibited the ability of PstI to cleave the genome at this site; in genomes in which ligation occurred, T4 DNA ligase was capable of covalently joining both modified and unmodified tetranucleotides to the gapped structures on both the 5' and the 3' ends with at least 90% efficiency. Evidence also is presented showing that the dG8-ABP-modified tetranucleotide was stable to the conditions of the recombinant DNA techniques used to insert it into the viral genome.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminobiphenyl Compounds↗
PubMed1986

Repair of oligodeoxynucleotides containing O6-methylguanine by O6-alkylguanine-DNA-alkyltransferase.

O6-Alkylguanine-DNA-alkyltransferase is a DNA repair protein known to carry out the transfer of alkyl groups from the O6-position of guanine in alkylated DNA to a cysteine acceptor site contained within its own protein sequence. We have examined the ability of this protein isolated from either E. coli or mammalian cells to perform this repair reaction in short oligodeoxynucleotides. Dodecadeoxynucleotides of the sequence 5'-dCGNGAATTCm6GCG-3' where N is any one of the normal four bases were all repaired very rapidly by the protein with 50% repair in less than 15 s at 0 degree C. The hexadeoxynucleotide 5'-dCGCm6GCG-3' was repaired slightly more slowly with 50% removal taking 7 min at 0 degree C and 1.5 min at 37 degrees C. The tetradeoxynucleotide 5'-dTm6GCA-3' was also a substrate but was repaired much more slowly requiring 45 min for 50% repair at 37 degrees C. These results indicate that (a) the AGT has a strong but not absolute preference for double-stranded DNA substrates; (b) the repair of O6-methylguanine is independent of the base opposite the lesion; and (c) that oligodeoxynucleotides as short as tetramers are substrates for repair by this protein.

DNA Repair↗
PubMed1986

Mutagenesis and repair of O6-substituted guanines.

The mutagenic activity of O6-methylguanine has been investigated using a single-stranded M13mp8 phage DNA molecule in which a single O6-methylguanine residue was positioned in the unique recognition site for the restriction endonuclease, Pst I. After introduction of this vector into Escherichia coli, progeny phage were produced, of which 0.4% were mutated in their Pst I site. To determine the impact of DNA repair on mutagenesis, levels of O6-methylguanine-DNA methyltransferase (an O6-methylguanine repair protein) were depleted in host cells by treatment with N-methyl-N'-nitro-N-nitrosoguanidine prior to viral DNA uptake. In these cells, the mutation frequency due to O6-methylguanine increased with increasing N-methyl-N'-nitro-N-nitrosoguanidine dose (the highest mutation frequency observed was 20%). DNA sequence analysis of mutant genomes revealed that O6-methylguanine induced G to A transitions, exclusively.

Coliphages↗
PubMed1985

Extrachromosomal probes for mutagenesis by carcinogens: studies on the mutagenic activity of O6-methylguanine built into a unique site in a viral genome.

This work examines the mutagenic activity of O6-methylguanine (O6MeGua), a DNA adduct formed by certain carcinogenic alkylating agents. A tetranucleotide, 5'-HOTpm6GpCpA-3', was synthesized and ligated into a four-base gap in the unique Pst I site of the duplex genome of the E. coli virus, M13mp8. The double-stranded ligation product was converted to single-stranded form and used to transform E. coli to produce progeny phage. The mutation frequency of O6MeGua was defined as the percentage of progeny phage with mutations in their Pst I site, and this value was determined to be 0.4%. To determine the impact of DNA repair on mutagenesis, cellular levels of O6MeGua-DNA methyltransferase (an O6MeGua-repair protein) were depleted by treatment of host cells for virus replication with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) prior to viral DNA uptake. In these host cells, the mutation frequency due to O6MeGua increased markedly with increasing MNNG dose (the highest mutation frequency observed was 20%). DNA sequence analysis of mutant genomes revealed that in both MNNG treated and untreated cells, O6MeGua induced exclusively G to A transitions.

Base Sequence↗
PubMed1985

Possible role for thymine glycol in the selective inhibition of DNA synthesis on oxidized DNA templates.

Single-stranded DNA of coliphage M13mp8 was treated with the oxidizing agent, KMnO4, under conditions that selectively form cis-5,6-dihydro-5,6-dihydroxythymine (thymine glycol). Treatment of DNA with 0.7 and 1.4 mM KMnO4 introduced approximately 200 and 400 thymine glycol residues, respectively, per genome. When these DNAs were used to transform Escherichia coli, it was observed that phage survival was reduced in a dose-dependent manner. In studies designed to investigate the effect of DNA oxidation products on replication in vitro, a complementary 15-mer oligodeoxynucleotide was annealed to the oxidized template and extended with the Klenow fragment of DNA polymerase I from E. coli. It was observed that lesions in oxidized DNA strongly inhibited DNA elongation and that DNA synthesis was stopped opposite thymine residues. This is taken as suggestive evidence that the thymine glycol is inhibitory to DNA replication.

Coliphages↗