DNA interstrand cross-links (ICLs) can be produced by reactions of DNA with a variety of endogenous metabolites (e.g. acrolein) and cancer chemotherapeutic agents (e.g. chloroethylnitrosoureas). ICLs are considered highly cytotoxic because they prevent separation of the two DNA strands required for DNA replication and transcription. Although not completely defined, the current models of ICLs repair pathways involve endonucleolytic incisions near the ICLs forming unhooked intermediates. In the replication-dependent ICL repair pathway, translesion DNA polymerases participate the bypass of these unhooked intermediates. Previously, acrolein derived minor grove ICLs formed between the exocyclic nitrogens of deoxyguanosines were used as a model to understand the lesion bypass capability of translesion DNA polymerases. The O6-guanine atom is reactive towards various alkylating agents. Bifunctional reagents such as 1,2-dibromoethane can react with the O6-guanine atom, forming DNA-protein cross-links in the presence of O6-alkylguanine-DNA alkyltransferase. We hypothesize that the O6-guanine DNA cross-link can serve as an important model to understand biological tolerance of the major groove DNA ICLs. We prepared site-specifically modified oligonucleotides containing an O6-guanine-butylene-O6-guanine DNA interstrand cross-link, potentially formed from an anti-cancer drug, 1,4-butanediol dimethanesulfonate. Two different sizes of DNA unhooked intermediates (i.e. 10-base and 4-base) were created mimicking products from nucleotide excision repair. Using these two model ICLs, we tested Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) and human DNA polymerase ? (pol ?) for translesion syntheses across the O6-guanine major groove ICLs. Primer extension assays showed that Dpo4 was very inefficient in bypassing the cross-links, whereas pol ? synthesized across the ICLs with moderate efficiencies. Blocking of the DNA replication was dependent on the size of the unhooked intermediate. Kinetic and structural studies are currently ongoing to understand the bypass mechanisms.

Translesion syntheses across O<sup>6</sup>-guanine-butylene-O<sup>6</sup>-guanine DNA interstrand cross-links

248th National Meeting (2014)

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Translesion syntheses across O6-guanine-butylene-O6-guanine DNA interstrand cross-links

Translesion syntheses across O⁶-guanine-butylene-O⁶-guanine DNA interstrand cross-links