Human Mitochondrial DNA Polymerase γ Exhibits Potential for Bypass and Mutagenesis at UV-induced Cyclobutane Thymine Dimers

Abstract

Cyclobutane thymine dimers (T-T) comprise the majority of DNA damage caused by short wavelength ultraviolet radiation. These lesions generally block replicative DNA polymerases and are repaired by nucleotide excision repair or bypassed by translesion polymerases in the nucleus. Mitochondria lack nucleotide excision repair, and therefore, it is important to understand how the sole mitochondrial DNA polymerase, pol γ, interacts with irreparable lesions such as T-T. We performed in vitro DNA polymerization assays to measure the kinetics of incorporation opposite the lesion and bypass of the lesion by pol γ with a dimer-containing template. Exonuclease-deficient pol γ bypassed thymine dimers with low relative efficiency; bypass was attenuated but still detectable when using exonuclease-proficient pol γ. When bypass did occur, pol γ misincorporated a guanine residue opposite the 3'-thymine of the dimer only 4-fold less efficiently than it incorporated an adenine. Surprisingly, the pol γ exonuclease-proficient enzyme excised the incorrectly incorporated guanine at similar rates irrespective of the nature of the thymines in the template. In the presence of all four dNTPs, pol γ extended the primer after incorporation of two adenines opposite the lesion with relatively higher efficiency compared with extension past either an adenine or a guanine incorporated opposite the 3'-thymine of the T-T. Our results suggest that T-T usually stalls mitochondrial DNA replication but also suggest a mechanism for the introduction of point mutations and deletions in the mitochondrial genomes of chronically UV-exposed cells.