Genotoxic effects of the major alkylation damage N7-methylguanine and methyl formamidopyrimidine.

Various alkylating agents are known to preferentially modify guanine in DNA, resulting in the formation of N7-alkylguanine (N7-alkylG) and the imidazole ring opened alkyl-formamidopyrimidine (alkyl-FapyG) lesions. Evaluating the mutagenic effects of N7-alkylG has been challenging due to the instability of the positively charged N7-alkylG. To address this issue, we developed a 2'-fluorine-mediated transition-state destabilization approach, which stabilizes N7-alkylG and prevents spontaneous depurination. We also developed a postsynthetic conversion of 2'-F-N7-alkylG DNA into 2'-F-alkyl-FapyG DNA. Using these methods, we incorporated site-specific N7-methylG and methyl-FapyG into pSP189 plasmid and determined their mutagenic properties in bacterial cells using the supF-based colony screening assay. The mutation frequency of N7-methylG was found to be less than 0.5%. Our crystal structure analysis revealed that N7-methylation did not significantly alter base pairing properties, as evidenced by a correct base pairing between 2'-F-N7-methylG and dCTP in Dpo4 polymerase catalytic site. In contrast, the mutation frequency of methyl-FapyG was 6.3%, highlighting the mutagenic nature of this secondary lesion. Interestingly, all mutations arising from methyl-FapyG in the 5'-GGT(methyl-FapyG)G-3' context were single nucleotide deletions at the 5'-G of the lesion. Overall, our results demonstrate that 2'-fluorination technology is a useful tool for studying the chemically labile N7-alkylG and alkyl-FapyG lesions.

Structure of the major oxidative damage 7,8-dihydro-8-oxoguanine presented into a catalytically competent DNA glycosylase.

If left unrepaired, the major oxidative DNA lesion 7,8-dihydro-8-oxoguanine (oxoG) promotes G-to-T transversions by favorably adopting a syn conformation and base pairing with dATP during replication. The human oxoG DNA glycosylase hOGG1 senses and removes oxoG amid millions-fold excess of guanine, thereby counteracting the genotoxic effects of the major oxidative damage. Crystal structures of hOGG1 in complex with oxoG-containing DNA have provided key insights into the lesion recognition and catalysis mechanisms of the enzyme. These lesion-recognition complex (LRC) structures typically involve a catalytically inactive hOGG1 mutant, where one of the catalytic-site amino acid residues is mutated to prevent the cleavage of oxoG. The use of a catalytically incompetent hOGG1 mutant has thus precluded understanding of unscathed interactions between oxoG and hOGG1 catalytic site as well as interactions among catalytic-site amino acid residues. As an orthogonal approach to visualize such interactions, we have co-crystallized a catalytically competent hOGG1 bound to 2'-fluoro-oxodG-containing DNA, a transition state destabilizing inhibitor that binds hOGG1 but is not processed by the enzyme. In this fluorinated lesion-recognition complex (FLRC), the 8-oxo moiety of oxoG is recognized by Gly42 and the Watson-Crick edge of oxoG is contacted by Gln315 and Pro266. The previously observed salt bridge between Lys249 and Cys253 is lacking in the FLRC, suggesting Lys249 is primed by Cys253 and poised for nucleophilic attack on C1' of oxodG. Overall, hOGG1 FLRC marks the first structure of oxoG presented into an intact catalytic site of hOGG1 and provides complementary insights into the glycosylase mechanisms of the enzyme.

Insights into the substrate discrimination mechanisms of methyl-CpG-binding domain 4.

G:T mismatches, the major mispairs generated during DNA metabolism, are repaired in part by mismatch-specific DNA glycosylases such as methyl-CpG-binding domain 4 (MBD4) and thymine DNA glycosylase (TDG). Mismatch-specific DNA glycosylases must discriminate the mismatches against million-fold excess correct base pairs. MBD4 efficiently removes thymine opposite guanine but not opposite adenine. Previous studies have revealed that the substrate thymine is flipped out and enters the catalytic site of the enzyme, while the estranged guanine is stabilized by Arg468 of MBD4. To gain further insights into the mismatch discrimination mechanism of MBD4, we assessed the glycosylase activity of MBD4 toward various base pairs. In addition, we determined a crystal structure of MBD4 bound to T:O6-methylguanine-containing DNA, which suggests the O6 and N2 of purine and the O4 of pyrimidine are required to be a substrate for MBD4. To understand the role of the Arg468 finger in catalysis, we evaluated the glycosylase activity of MBD4 mutants, which revealed the guanidinium moiety of Arg468 may play an important role in catalysis. D560N/R468K MBD4 bound to T:G mismatched DNA shows that the side chain amine moiety of the Lys stabilizes the flipped-out thymine by a water-mediated phosphate pinching, while the backbone carbonyl oxygen of the Lys engages in hydrogen bonds with N2 of the estranged guanine. Comparison of various DNA glycosylase structures implies the guanidinium and amine moieties of Arg and Lys, respectively, may involve in discriminating between substrate mismatches and nonsubstrate base pairs.