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 eta.
Xeroderma pigmentosum variant (XPV) patients carry germ-line mutations in DNA polymerase eta (poleta), 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 poleta-proficient and deficient human cells. TLS across the TT CPD lesion was reduced by 2.6-4.4-fold in cells lacking poleta, and exhibited a strong 6-17-fold increase in mutation frequency at the TT CPD. All targeted mutations (74%) in poleta-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 poleta, 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 poleta-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 poleta. 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 poleta in error-free TLS across CPD in human cells, and suggest a potential involvement, although minor, of poleta in TLS across 6-4 PP under some conditions.
Hendel
Ayal
A
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Ziv
Omer
O
Gueranger
Quentin
Q
Geacintov
Nicholas
N
Livneh
Zvi
Z
D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000172:deficiency* / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D011740:Pyrimidine Dimers / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014983:Xeroderma Pigmentosum / Q000201:enzymology; D000099270:Y-Family DNA Polymerases
D004247:DNA; D004259:DNA-Directed DNA Polymerase; D011740:Pyrimidine Dimers; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases