Abnormal, error-prone bypass of photoproducts by xeroderma pigmentosum variant cell extracts results in extreme strand bias for the kinds of mutations induced by UV light

Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a gre atly increased susceptibility to sunlight-induced skin cancer. Cells from t he majority of patients are defective in nucleotide excision repair. Howeve r, cells from one set of patients, XP variants, exhibit normal repair but a re abnormally slow in replicating DNA containing UV photoproducts, The freq uency of UV radiation-induced mutations in the XP variant cells is signific antly higher than that in normal human cells, Furthermore, the kinds of UV- induced mutations differ very significantly from normal. Instead of transit ions, mainly C-->T, 30% of the base substitutions consist of C-->A transver sions, all arising from photoproducts located in one strand. Mutations invo lving cytosine in the other strand are almost all C-->T transitions, Forty- five percent of the substitutions involve thymine, and the majority are tra nsversions, To test the hypothesis that the UV hypermutability and the abno rmal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells,vith those from HeLa cells and a fibroblast cell strain, MSU-1.2 for the ability to replicate a UV irr adiated form I M13 phage. The M13 template contains a simian virus 40 origi n of replication located directly to the left or to the right of the target gene, lacZ alpha, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to similar to 37% of the control value required only 1 photoproduct per template for XP varia nt cell extracts, but similar to 2.2 photoproducts far HeLa or MSU-1.2 cell extracts, The frequency of mutants induced was four times higher with XP v ariant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP varia nt cell extracts, the proportion of C-->A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the te mplate for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, th is value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, an d virtually all were T-->A transversions. Sequence analysis of the coding r egion of the catalytic subunit of DNA polymerase delta in XP variant cell l ines revealed two polymorphisms, but these do not account for the reduced b ypass fidelity, Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cell s, bypass of photoproducts involving cytosine in the template for the leadi ng strand differs significantly from that of photoproducts in the lagging s trand.