Reduced efficiency and increased mutagenicity of translesion DNA synthesis across a TT cyclobutane pyrimidine dimer, but not a TT 6-4 photoproduct, in human cells lacking DNA polymerase η

Abstract

Xeroderma pigmentosum variant (XPV) patients carry germ-line mutations in DNA polymerase η (polη), a major translesion DNA synthesis (TLS) polymerase, and exhibit severe sunlight sensitivity and high predisposition to skin cancer. Using a quantitative TLS assay system based on gapped plasmids we analyzed TLS across a site-specific TT CPD (thymine–thymine cyclobutane pyrimidine dimer) or TT 6-4 PP (thymine–thymine 6-4 photoproduct) in three pairs of polη-proficient and deficient human cells. TLS across the TT CPD lesion was reduced by 2.6–4.4-fold in cells lacking polη, and exhibited a strong 6–17-fold increase in mutation frequency at the TT CPD. All targeted mutations (74%) in polη-deficient cells were opposite the 3′T of the CPD, however, a significant fraction (23%) were semi-targeted to the nearest nucleotides flanking the CPD. Deletions and insertions were observed at a low frequency, which increased in the absence of polη, consistent with the formation of double strand breaks due to defective TLS. TLS across TT 6-4 PP was about twofold lower than across CPD, and was marginally reduced in polη-deficient cells. TLS across TT 6-4 PP was highly mutagenic (27–63%), with multiple mutations types, and no significant difference between cells with or without polη. Approximately 50% of the mutations formed were semi-targeted, of which 84–93% were due to the insertion of an A opposite the template G 5′ to the 6-4 PP. These results, which are consistent with the UV hyper-mutability of XPV cells, highlight the critical role of polη in error-free TLS across CPD in human cells, and suggest a potential involvement, although minor, of polη in TLS across 6-4 PP under some conditions.