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

DNA Repair (Amst). 2008 Oct 1;7(10):1636-46. doi: 10.1016/j.dnarep.2008.06.008. Epub 2008 Aug 3.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • DNA / biosynthesis*
  • DNA-Directed DNA Polymerase / deficiency*
  • DNA-Directed DNA Polymerase / metabolism
  • Fibroblasts / enzymology*
  • Fibroblasts / pathology
  • Humans
  • Mutagenesis*
  • Plasmids / genetics
  • Pyrimidine Dimers / metabolism*
  • Sequence Analysis, DNA
  • Xeroderma Pigmentosum / enzymology


  • Pyrimidine Dimers
  • DNA
  • DNA-Directed DNA Polymerase
  • Rad30 protein