Polymerase zeta contributes to mutation signature arising from alkylating agent exposure

Exogenous and endogenous harmful agents are constantly damaging DNA. Certain chemicals have the capability to bind covalently to DNA forming DNA adducts. The resulting unnatural base cannot be processed by usual replicative polymerase during S phase. Thanks to translesion polymerases such as polymerase ζ (polζ), DNA adducts can be bypassed but often at the cost of a wrong base being inserted opposite the damage. Polζ is known to be involved in damage-induced mutations because of its great efficiency to extend DNA from a mismatch. We are interested in the mutagenesis role of polζ in the bypass of O6-methylguanine adduct (O6-MeG). We first adopted an enzyme-based approach by using the purified version of human polζ and a DNA template containing O6-MeG damage. We performed enzyme kinetic studies to look at the base inserted opposite O6-MeG and how efficiently polζ extends from different mismatches. Polζ was found to insert in a low extend a T opposite O6-MeG, and very efficiently extend from that mismatch. Our hypotheses is that polζ is giving rise to G > A transversions in the bypass of O6-MeG, this process being influenced by the sequence context. To add more biological relevance we want to use mouse embryonic fibroblasts (MEFs) deficient in REV3L, the catalytic subunit of polζ. WT and REV3L-/- MEFs will be treated with an alkylating agent to generate O6-MeG. Whole-genome sequencing of single clones will be performed in order to assess the role of polζ in mutagenesis following alkylating agent exposure