Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase

3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glycosylase not only for the cytotoxic 3MeA DNA lesion, but also for the mutagenic 1,N6-ethenoadenine (epsilonA) and hypoxanthine lesions. Aag appears to be the only 3MeA and hypoxanthine DNA glycosylase in liver, testes, kidney, and lung, and the only epsilonA DNA glycosylase in liver, testes, and kidney; another epsilonA DNA glycosylase may be expressed in lung. Although alkyladenine DNA glycosylase has the capacity to remove 8-oxoguanine DNA lesions, it does not appear to be the major glycosylase for 8-oxoguanine repair. Fibroblasts derived from Aag -/- mice are alkylation sensitive, indicating that Aag -/- mice may be similarly sensitive.     

Correlation between sequence-dependent glycosylase repair and the thermal stability of oligonucleotide duplexes containing 1, N6-ethenoadenine

Previous experiments on DNA sequence context reported that base modification, replication, and repair are affected by the nature of neighbor bases. We now report that repair by mammalian alkylpurine-DNA-N-glycosylases (APNG) of 15-mer oligonucleotides with a central 1,N6-ethenoadenine (epsilonA), flanked by 5' and 3' tandem bases, is also highly sequence dependent. Oligonucleotides with the central sequences -GGepsilonAGG- or -CCepsilonACC- are repaired 3-5-fold more efficiently than those containing -AAepsilonAAA- or -TTepsilonATT- when using human or mouse APNG. Melting curves of the same duplexes showed that oligomers with G.C/C. G neighbors were less denatured than those with A.T/T.A neighbors at 37 degreesC. This sequence-dependent difference in denaturation correlates with the relative thermodynamic stability of oligomers with G.C/C.G or A.T/T.A neighbors. The dependence of repair on thermal stability was confirmed by enzyme reactions performed over 0-45 degreesC. Under these conditions, repair of epsilonA flanked by G.C/C.G was dramatically increased at 37 degreesC with continuous increase up to 45 degreesC, in contrast to that with flanking A.T/T. A pairs, which was in agreement with the degree of denaturation of these duplexes. These results indicate that the thermodynamic stability conferred by base pairs flanking epsilonA plays an essential role in maintaining the integrity of the duplex structure which is necessary for repair.     

Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site

Reactive oxygen species produce different lesions in DNA. Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis. This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1). Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein. It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion. However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype.     

Detection of 1,N6-ethenoadenine in rat urine after chloroethylene oxide exposure

The four etheno adducts of vinyl chloride formed in DNA, 1,N6-ethenoadenine (epsilonA), 3,N4-ethenocytosine, 1,N2-ethenoguanine and N2,3-ethenoguanine were previously reported to be released from DNA by a family of enzymes in the base-excision repair pathway (Dosanjh et al., Proc. Natl Acad. Sci. USA, 91, 1024-1028, 1994; Hang et al., Carcinogenesis, 17, 155-157, 1996; Hang et al., Proc. Natl Acad. Sci. USA, 94, 12869-12874, 1997). Adducts excised from DNA by glycosylases are usually excreted in urine and have been reported to be potential biomarkers of DNA damage in exposed individuals. In this study, we report the detection of epsilonA in the urine of rats exposed to chloroethylene oxide (CEO) using immunoaffinity columns made with specific monoclonal antibodies for enrichment, followed by quantitation by HPLC with fluorescence detection. Chemical analysis of urine samples revealed the presence of a compound chromatographically identical to authentic epsilonA standard. This compound was confirmed by mass spectral analysis. EpsilonA was present in urine of control and CEO-treated rats, with the latter having up to 50-fold greater amounts. The cumulative excretion of epsilonA reached a plateau between 24 and 48 h post-exposure. While it is clear that CEO treatment results in increased excretion of epsilonA, the exact source of the adduct is unknown. When rats were administered epsilonA i.v., approximately 10% of the administered dose was excreted in urine. This research demonstrates that urinary excretion of epsilonA may be a potential biomarker for in vivo alkylation of DNA and nucleotide pools.     

An oxidative damage-specific endonuclease from rat liver mitochondria

Reactive oxygen species have been shown to generate mutagenic lesions in DNA. One of the most abundant lesions in both nuclear and mitochondrial DNA is 7,8-dihydro-8-oxoguanine (8-oxoG). We report here the partial purification and characterization of a mitochondrial oxidative damage endonuclease (mtODE) from rat liver that recognizes and incises at 8-oxoG and abasic sites in duplex DNA. Rat liver mitochondria were purified by differential and Percoll gradient centrifugation, and mtODE was extracted from Triton X-100-solubilized mitochondria. Incision activity was measured using a radiolabeled double-stranded DNA oligonucleotide containing a unique 8-oxoG, and reaction products were separated by polyacrylamide gel electrophoresis. Gel filtration chromatography predicts mtODE's molecular mass to be between 25 and 30 kDa. mtODE has a monovalent cation optimum between 50 and 100 mM KCl and a pH optimum between 7.5 and 8. mtODE does not require any co-factors and is active in the presence of 5 mM EDTA. It is specific for 8-oxoG and preferentially incises at 8-oxoG:C base pairs. mtODE is a putative 8-oxoG glycosylase/lyase enzyme, because it can be covalently linked to the 8-oxoG oligonucleotide by sodium borohydride reduction. Comparison of mtODE's activity with other known 8-oxoG glycosylases/lyases and mitochondrial enzymes reveals that this may be a novel protein.     

Role of lysine-57 in the catalytic activities of Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein)

The Escherichia coli Fpg protein is involved in the repair of oxidized residues. We examined, by targeted mutagenesis, the effect of the conserved lysine residue at position 57 upon the various catalytic activities of the Fpg protein. Mutant Fpg protein with Lys-57-->Gly (K57G) had dramatically reduced DNA glycosylase activity for the excision of 7,8-dihydro-8-oxo-guanine (8-oxoG). While wild type Fpg protein cleaved 8-oxoG/C DNA with a specificity constant ( k cat/ K M) of 0.11/(nM@min), K57G cleaved the same DNA 55-fold less efficiently. FpgK57G was poorly effective in the formation of Schiff base complex with 8-oxoG/C DNA. The efficiency in the binding of 8-oxoG/C DNA duplex for K57G mutant was decreased 16-fold. The substitution of Lys-57 for another basic amino acid Arg (K57R) had a slight effect on the 8-oxoG-DNA glycosylase activity and Schiff base formation. The DNA glycosylase activities of FpgK57G and FpgK57R using 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine residues as substrate were comparable to that of wild type Fpg. In vivo, the mutant K57G, in contrast to the mutant K57R and wild type Fpg, only partially restored the ability to prevent spontaneously induced transitions G/C-->T/A in E.coli BH990 ( fpg mutY ) cells. These results suggest an important role for Lys-57 in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo.     

Chronic thromboembolic pulmonary hypertension: hemodynamic effects of selective pulmonary artery DSA with nonionic contrast media]

This paper examines the relationship in Escherichia coli between the in vivo content of 8-oxoguanine (8-oxoG) in chromosomal DNA and deficiencies of various key antioxidant defences. The structural genes for catalases (katG and katE), cytosolic superoxide dismutases (sodA and sodB) or formamidopyrimidine-DNA glycosylase (fpg) were inactivated to obtain bacterial strains lacking the scavenger enzymes for H2O2 or O2.- or the DNA repair protein for 8-oxoG. Wild-type bacteria showed 5-fold increased sensitivity to both lethality and mutagenesis by H2O2 in K medium (1% casamino acids and 1% glucose), as compared with nutrient broth. This higher sensitivity was associated with increased chromosomal oxidative damage, estimated as the 8-oxodG content, and with a marked decrease in both catalase and SOD activities. Bacteria lacking both cytosolic SODs (sodA sodB mutant) displayed increased 8-oxodG content in chromosomal DNA (2.8-fold that of the wild-type) when grown under standard aerated conditions. Comparatively, no significant difference in 8-oxodG content was observed in cells grown without aeration. Bacteria totally devoid of catalase activity (katG katE mutant) showed wild-type contents of 8-oxodG in chromosomal DNA when grown under aerated conditions. Nevertheless, the protective role of catalase in preventing formation of 8-oxodG in chromosomal DNA became evident under oxidative stress conditions: growth under hyperoxygenation and, particularly, following H2O2 exposure. Catalase deficiency resulted in a dramatic decrease in viability after H2O2 exposure. A deficiency of Fpg protein also sensitized E.coli to H2O2 lethality, though to lesser extent than a deficiency of catalase activity. However, the scavenger enzyme and the DNA repair protein protected equally against 8-oxoG formed in vivo upon H2O2 treatment.     

Role of mismatch repair in the Escherichia coli UVM response

Mutagenesis at 3,N4-ethenocytosine (epsilonC), a nonpairing mutagenic lesion, is significantly enhanced in Escherichia coli cells pretreated with UV, alkylating agents, or H2O2. This effect, termed UVM (for UV modulation of mutagenesis), is distinct from known DNA damage-inducible responses, such as the SOS response, the adaptive response to alkylating agents, or the oxyR-mediated response to oxidative agents. Here, we have addressed the hypothesis that UVM results from transient depletion of a mismatch repair activity that normally acts to reduce mutagenesis. To test whether the loss of mismatch repair activities results in the predicted constitutive UVM phenotype, E. coli cells defective for methyl-directed mismatch repair, for very-short-patch repair, or for the N-glycosylase activities MutY and MutM were treated with the UVM-inducing agent 1-methyl-3-nitro-1-nitrosoguanidine, with subsequent transfection of M13 viral single-stranded DNA bearing a site-specific epsilonC lesion. Survival of the M13 DNA was measured as transfection efficiency, and mutation fixation at the lesion was characterized by multiplex sequencing technology. The results showed normal UVM induction patterns in all the repair-defective strains tested. In addition, normal UVM induction was observed in cells overexpressing MutH, MutL, or MutS. All strains displayed UVM reactivation, the term used to describe the increased survival of epsilonC-containing DNA in UVM-induced cells. Taken together, these results indicate that the UVM response is independent of known mismatch repair systems in E. coli and may thus represent a previously unrecognized misrepair or misreplication pathway.     

In search of a mutational hotspot

In vitro selection was used to define sequence contexts that significantly enhanced the mutagenic potential of 7, 8-dihydro-8-oxoguanine (8-oxoG). Contexts that simultaneously reduced the efficiency of 8-oxoG cleavage by formamidopyrimidine DNA N-glycosylase and increased the efficiency of misincorporating A opposite the lesion by DNA polymerase were isolated from a pool of 4(8) random octanucleotide sequences. Kinetic analysis showed that the combined effects of poor repair and high miscoding resulted in 10(2)- to 10(3)-fold increase in the mutagenic potential of 8-oxoG. Furthermore, the isolated sequence contexts correlated strongly with G --> T transversion hotspots in spontaneous mutational spectra reported for the Escherichia coli lacI and human p53 and factor IX genes. We present an example directly linking the interplay between DNA repair and replication to a "high risk sequence" for base substitution.     

Characterization of the recombinant MutY homolog, an adenine DNA glycosylase, from yeast Schizosaccharomyces pombe

The mutY homolog (SpMYH) gene from a cDNA library of Schizosaccharomyces pombe encodes a protein of 461 amino acids that displays 28 and 31% identity to Escherichia coli MutY and human MutY homolog (MYH), respectively. Expressed SpMYH is able to complement an E. coli mutY mutant to reduce the mutation rate. Similar to E. coli MutY protein, purified recombinant SpMYH expressed in E. coli has adenine DNA glycosylase and apurinic/apyrimidinic lyase activities on A/G- and A/7,8-dihydro-8-oxoguanine (8-oxoG)-containing DNA. However, both enzymes have different salt requirements and slightly different substrate specificities. SpMYH has greater glycosylase activity on 2-aminopurine/G and A/2-aminopurine but weaker activity on A/C than E. coli MutY. Both enzymes also have different substrate binding affinity and catalytic parameters. Although SpMYH has great affinity to A/8-oxoG-containing DNA as MutY, the binding affinity to A/G-containing DNA is substantially lower for SpMYH than MutY. SpMYH has similar reactivity to both A/G- and A/8-oxoG-containing DNA; however, MutY cleaves A/G-containing DNA about 3-fold more efficiently than it does A/8-oxoG-containing DNA. Thus, SpMYH is the functional eukaryotic MutY homolog responsible for reduction of 8-oxoG mutational effect.     

Sequence context is an important determinant in the mutagenic potential of 1,N6-ethenodeoxyadenosine (epsilonA): formation of epsilonA basepairs and elongation in defined templates

Many laboratories have obtained data on mutagenicity of modified bases in naturally occurring DNA sequences. It has often been noted that mutation is favored in certain sequence contexts, sometimes termed 'hot spots'. This approach to the contribution of neighboring sequences does not permit a systematic study of both the qualitative and quantitative mutational frequencies. In the present experiments we have chosen to use the exocyclic adduct, 1,N6-etheno A (epsilonA), site-specifically placed in a defined 25-mer oligonucleotides in which epsilonA is flanked by differing 5' and 3' tandem bases. Mutation was assessed using an in vitro replication assay and five polymerases of varying fidelity. The relevant central sequences were 3' --> 5' -CC-epsilonA-CC-, -GG-epsilonA-GG-, -TT-epsilonA-TT-, -AA-epsilonA-AA-, -GG-epsilonA-TT-, -TT-epsilonA-AA-, -AT-epsilonA-TT- and -TA-epsilonA-TA-. Using the Klenow fragment (Kf) (exo+ or exo-) of E. coli Pol I, it was found the epsilonA is an ambiguous base and, with varying efficiencies, all four dNTPs could be inserted opposite epsilonA in all sequences. However, only 3' --> 5' -TT-epsilonA-TT-, -GG-epsilonA-TT- and -AT-epsilonA-TT- were fully extended to a significant extent. The only sequences essentially blocked at the position of epsilonA were -AA-epsilonA-AA- and -TT-epsilonA-AA-. The others were intermediate. When replication was performed with Sequenase, MMLV RT or HIV RT, different patterns were observed, in which replication terminated one base prior to epsilonA, at epsilonA, or one base after epsilonA without further extension. In favored sequences, using the Klenow fragment, an epsilonA x N pair could be extended to form normal basepairs. No extension could be demonstrated in sequences in which tandem adenines were 5' to epsilonA. Kinetic data showed that two of the epsilonA x N pairs, epsilonA x A and epsilonA x C, could form at 10 microM or less dNTP. Which bases were preferentially inserted opposite epsilonA was a function of the flanking bases. Under the kinetic conditions used, epsilonA x T did not form even at 1 mM dTTP. These results indicate that the chemical structure of an adduct is not the only determinant of mutagenic efficiency. It is likely that the effect of the adduct on replication is due to the changes in the structural environment conferred by the flanking bases.     

Identification of a poxvirus gene encoding a uracil DNA glycosylase

An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 RNA polymerase promoter. A highly specific ethidium bromide fluorescence assay of the in vitro translation product determined that the encoded protein does indeed possess uracil DNA glycosylase activity. Open reading frames from other poxviruses, including vaccinia virus (HindIII D4R) and fowlpox (D4), are highly homologous to D6R of SFV and are predicted to encode uracil DNA glycosylases. Identification of the SFV uracil DNA glycosylase provides evidence that this poxviral protein is involved in the repair of the viral DNA genome. Since this enzyme performs only the initial step required for the removal of uracil from DNA, creating an apyrimidinic site, we suggest that other, possibly virus-encoded, repair activities must be present in the cytoplasm of infected cells to complete the uracil excision repair pathway.     

Induction of topoisomerase I cleavage complexes by the vinyl chloride adduct 1,N6-ethenoadenine

We used purified mammalian topoisomerases I (top1) and oligonucleotides to study top1-mediated cleavage and religation in the presence of a potent carcinogenic adduct, 1,N6-ethenoadenosine (epsilonA) incorporated immediately downstream of a unique top1 cleavage site. We found tha epsilonA markedly enhanced top1 cleavage complexes when it was incorporated at the +1 position of the top1 cleavage. This enhancement was due to a reduction of the religation step of the top1 reaction. In addition, epsilonA reduced the top1-mediated cleavage and decreased binding of the enzyme to DNA. We also studied the effects of the epsilonA adduct on top1 trapping by camptothecin (CPT), a well known top1 inhibitor. CPT was inactive when epsilonA was present at the +1 position. Alkylation of the top1 cleavage complex by 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT) was also blocked by the epsilonA adduct. Altogether, these results demonstrate that the epsilonA carcinogenic adduct can efficiently trap human top1 and mimic CPT effects. Normal hydrogen bonding of the base pairs immediately downstream from the top1 cleavage site is probably essential for efficient DNA religation and binding of camptothecins in the top1 cleavage complex.     

Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-->C:G transversions

Low rates of spontaneous G:C-->C:G transversions would be achieved not only by the correction of base mismatches during DNA replication but also by the prevention and removal of oxidative base damage in DNA. Escherichia coli must have several pathways to repair such mismatches and DNA modifications. In this study, we attempted to identify mutator loci leading to G:C-->C:G transversions in E.coli. The strain CC103 carrying a specific mutation in lacZ was mutagenized by random miniTn 10 insertion mutagenesis. In this strain, only the G:C-->C:G change can revert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose. Mutator strains were detected as colonies with significantly increased rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal. We screened approximately 40 000 colonies and selected several mutator strains. The strain GC39 showed the highest mutation rate to Lac+. The gene responsible for the mutator phenotypes, mut39 , was mapped at around 67 min on the E.coli chromosome. The sequencing of the miniTn 10 -flanking DNA region revealed that the mut39 was identical to the mutY gene of E.coli. The plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains. Purified MutY protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine (8-oxoG):G and 8-oxoG:A. Furthermore, we found that the MutY protein had a DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G mispair. These results demonstrate that the MutY protein prevents the generation of G:C-->C:G transversions by removing guanine from the 8-oxoG:G mispair in E.coli.     

All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase

We have previously reported that human cells and tissues contain a 1,N6-ethenoadenine (epsilon A) binding protein, which, through glycosylase activity, releases both 3-methyladenine (m3A) and epsilon A from DNA treated with methylating agents or the vinyl chloride metabolite chloroacetaldehyde, respectively. We now find that both the partially purified human epsilon A-binding protein and cell-free extracts containing the cloned human m3A-DNA glycosylase release all four cyclic etheno adducts--namely epsilon A, 3,N4-ethenocytosine (epsilon C), N2,3-ethenoguanine (N2,3-epsilon G), and 1,N2-ethenoguanine (1,N2-epsilon G). Base release was both time and protein concentration dependent. Both epsilon A and epsilon C were excised at similar rates, while 1,N2-epsilon G and N2,3-epsilon G were released much more slowly under identical conditions. The cleavage of glycosyl bonds of several heterocyclic adducts as well as those of simple methylated adducts by the same human glycosylase appears unusual in enzymology. This raises the question of how such a multiple, divergent activity evolved in humans and what may be its primary substrate.     

The critical active-site amine of the human 8-oxoguanine DNA glycosylase, hOgg1: direct identification, ablation and chemical reconstitution

BACKGROUND: Base-excision DNA repair (BER) is the principal pathway responsible for the removal of aberrant, genotoxic bases from the genome and restoration of the original sequence. Key components of the BER pathway are DNA glycosylases, enzymes that recognize aberrant bases in the genome and catalyze their expulsion. One major class of such enzymes, glycosylase/lyases, also catalyze scission of the DNA backbone following base expulsion. Recent studies indicate that the glycosylase and lyase functions of these enzymes are mechanistically unified through a common amine-bearing residue on the enzyme, which acts as both the electrophile that displaces the aberrant base and an electron sink that facilitates DNA strand scission through imine (Schiff base)/conjugate elimination chemistry. The identity of this critical amine-bearing residue has not been rigorously established for any member of a superfamily of BER glycosylase/lyases. RESULTS: Here, we report the identification of the active-site amine of the human 8-oxoguanine DNA glycosylase (hOgg1), a human BER superfamily protein that repairs the mutagenic 8-oxoguanine lesion in DNA. We employed Edman sequencing of an active-site peptide irreversibly linked to substrate DNA to identify directly the active-site amine of hOgg1 as the epsilon-NH2 group of Lys249. In addition, we observed that the repair-inactive but recognition-competent Cys249 mutant (Lys249-->Cys) of hOgg1 can be functionally rescued by alkylation with 2-bromoethylamine, which functionally replaces the lysine residue by generating a gamma-thia-lysine. CONCLUSIONS: This study provides the first direct identification of the active-site amine for any DNA glycosylase/lyase belonging to the BER superfamily, members of which are characterized by the presence of a helix-hairpin-helix-Gly/Pro-Asp active-site motif. The critical lysine residue identified here is conserved in all members of the BER superfamily that exhibit robust glycosylase/lyase activity. The ability to trigger the catalytic activity of the Lys249-->Cys mutant of hOgg1 by treatment with the chemical inducer 2-bromoethylamine may permit snapshots to be taken of the enzyme acting on its substrate and could represent a novel strategy for conditional activation of catalysis by hOgg1 in cells.     

Solution structure of a DNA duplex containing the exocyclic lesion 3,N4-etheno-2'-deoxycytidine opposite 2'-deoxyguanosine

Vinyl chloride reacts with cellular DNA producing 3,N4-etheno-2'-deoxycytidine (epsilonC) along with other exocyclic adducts. The solution structure of an oligodeoxynucleotide duplex containing an epsilonC.dG base pair was determined by high-resolution NMR spectroscopy and molecular dynamics simulations. NMR data indicated that the duplex adopts a right-handed helical structure having all residues in anti orientation around the glycosylic torsion angle. The epsilonC adduct has a sugar pucker in the C3'-endo/C4'-exo region while the rest of the residues are in the C2'-endo/C3'-exo range. NOE interactions established Watson-Crick alignments for canonical base pairs of the duplex. The imino proton of the lesion-containing base pair resonated as a sharp signal that was resistant to water exchange, suggesting hydrogen bonding. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. The refined structures are slightly bent at the lesion site without major perturbations of the sugar-phosphate backbone. The adduct is displaced and shifted toward the major groove of the helix while its partner on the complementary strand remains stacked. The epsilonC(anti).dG(anti) base pair alignment is sheared and stabilized by the formation of hydrogen bonds. The biological implications of structures of epsilonC-containing DNA duplexes are discussed.     

Incision at DNA G.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.T binding reaction

We have investigated the specificities of G.T mismatch binding proteins and of G.T mismatch cleavage in extracts of mammalian cells. G.T mismatch-specific protein:DNA complex formation by cell extracts was independent of the local sequence context of the mismatch. Cell extracts performed similar levels of protein binding to DNA substrates in which a single G.T mispair was preceded by T, G, A, C, or 5-meC. In contrast, incision by extracts of the T-containing strand of a G.T mismatch exhibited a strong sequence specificity and efficient strand cleavage was only observed when the mismatched G was in a CpG sequence. Thus, oligonucleotides containing either CpgGpT or 5meCpGGpT were efficiently incised, but not those containing GpGCpT, ApGTpT, or TpGApT sequences. Cell lines made resistant to the alkylating agent N-methyl-N-nitrosourea have previously been found to be defective in a G.T mismatch binding reaction. The defect in binding by extracts prepared from these cells extended to G.T mismatches in several sequence contexts. The variant extracts nevertheless incised G.T mismatches normally suggesting that this particular binding activity is not required for incision. The data indicate that incision by this activity is targeted to the CpG sequences in which G.T mismatches are formed by the mutagenic deamination of DNA 5-methylcytosine. In this regard the repair pathway resembles the very short patch (vsp) repair pathway in Escherichia coli.     

DNA mismatch repair catalyzed by extracts of mitotic, postmitotic, and senescent Drosophila tissues and involvement of mei-9 gene function for full activity

Extracts of Drosophila embryos and adults have been found to catalyze highly efficient DNA mismatch repair, as well as repair of 1- and 5-bp loops. For mispairs T.G and G.G, repair is nick dependent and is specific for the nicked strand of heteroduplex DNA. In contrast, repair of A.A, C.A, G.A, C.T, T.T, and C.C is not nick dependent, suggesting the presence of glycosylase activities. For nick-dependent repair, the specific activity of embryo extracts was similar to that of extracts derived from the entirely postmitotic cells of young and senescent adults. Thus, DNA mismatch repair activity is expressed in Drosophila cells during both development and aging, suggesting that there may be a function or requirement for mismatch repair throughout the Drosophila life span. Nick-dependent repair was reduced in extracts of animals mutant for the mei-9 gene. mei-9 has been shown to be required in vivo for certain types of DNA mismatch repair, nucleotide excision repair (NER), and meiotic crossing over and is the Drosophila homolog of the yeast NER gene rad1.