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Mismatched T:G base pairs can arise during de novo replication as well as base excision repair (BER). In particular, the action of the gap-filling polymerase β (Polβ) can generate a T:G pair as well as a nick in the DNA backbone. The processing of a nicked T:G mispair is poorly understood. We are interested in understanding whether the T:G-specific DNA glycosylase MBD4 can recognize and process nicked T:G mismatches. We have discovered that MBD4 binds a nicked T:G-containing DNA, but does not cleave thymine opposite guanine. To gain insight into this, we have determined a crystal structure of human MBD4 bound to a nicked T:G-containing DNA. This structure displayed the full insertion of thymine into the catalytic site and the recognition of thymine based on the catalytic site’s amino acid residues. However, thymine excision did not occur, presumably due to the inactivation of the catalytic D560 carboxylate nucleophile via a polar interaction with the 5′-hydrogen phosphate of the nicked DNA. The nicked complex was greatly stabilized by an ordered water molecule that formed four hydrogen bonds with the nicked DNA and MBD4. Interestingly, the arginine finger R468 did not engage in the phosphate pinching that is commonly observed in T:G mismatch recognition complex structures. Instead, the guanidinium moiety of R468 made bifurcated hydrogen bonding interactions with O6 of guanine, thereby stabilizing the estranged guanine. These observations suggest that R468 may sense and disrupt T:G pairs within the DNA duplex and stabilize the flipped-out thymine. The structure described here would be a close mimic of an intermediate in the base extrusion pathway induced by DNA glycosylase.

DNA interstrand cross-links (ICLs) are among the most cytotoxic forms of DNA damage, arising when the two strands of the DNA helix are covalently linked by crosslink-inducing agents such as bifunctional alkylating agents and reactive aldehydes. Several studies have demonstrated that ICLs can also be induced by reactive oxygen and nitrogen species. We previously reported that under oxidative conditions, the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine (oxoA) can efficiently generate a novel class of oxoA-G ICLs, structurally resembling guanine–guanine (G–G) cross-links that can be induced by reactive nitrogen species. To investigate the mutagenic potential of these oxidation-induced ICLs in cells, we employed a SupF-based mutagenesis assay using bacterial cells. A single site-specific oxoA–G ICL was synthesized and incorporated into a plasmid, which was then introduced into an E. coli reporter strain to assess mutation profiles induced by both oxoA and oxoA–G ICLs. Our results show that oxoA–G ICLs cause A-to-C/T and G-to-C transversion mutations at the oxoA-G cross-link site, demonstrating highly promutagenic nature of the lesion in bacterial cells. We propose that the oxoA–G ICL may promote transversion mutations, likely driven by a syn conformer of unhooked oxoA-G ICL repair intermediates during translesion synthesis.