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Xeroderma pigmentosum (XP) variant patients are genetically predisposed to sunlight-induced skin cancer.Fibroblasts derived from these patients are extremely sensitive to the mutagenic effect of UV radiation and are abnormally slow in replicating DNA containing UV-induced photoproducts. However, unlike cells from the majority of XP patients, XP variant cells have a normal or nearly normal rate of nucleotide excision repair of such damage. To determine whether their IUV hypermutability reflected a slower rate of excision of photoproducts specifically during early S phase when the target gene for mutations, i.e., the hypoxanthine (guanine) phosphoribosyltransferase gene (HPRT), is replicated, we synchronized diploid populations of normal and XP variant fibroblasts, irradiated them in eariy S phase, and compared the rate of loss of cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidones from DNA during S phase. There was no difference. Both removed 94% of the 6-4 pyrimidine-pyrimidones within 8 h and 40%o of the dimers within 11 h. There was also no difference between the two cell lines in the rate of repair during G1 phase. To determine whether the hypermutability resulted from abnormal error-prone replication of DNA containing photoproducts, we determined the spectra of mutations induced in the coding region of the HPRT gene of XP variant cells irradiated in eariy S and G1 phases and compared them with those found in normal cells. The majority of the mutations in both types of cells were base substitutions, but the two types of cells differed significantly from each other in the kinds of substitutions observed either in mutants from S phase (P < 0.01) or from G, phase (P = 0.03). In the variant cells, the substitutions were mainly transversions (58% in S, 73% in G1). In normal cells, transversions were much rarer (8% in S, 24% in G1; P < 0.001 for S, P < 0.01 for G1). In the normal cells irradiated in S, the majority of the substitutions were G.C-*A.T, and most involved CC photoproducts in the transcribed strand. In the variant cells irradiated in S, substitutions involving cytosine in the transcribed strand were G.C--T.A transversions exclusively. G.C-oA.T transitions made up a much smaller fraction of the substitutions than in normal cells (P < 0.02), and all of them involved photoproducts located in the nontranscribed strand. The data strongly suggest that XP The hypermutability cannot be accounted for by errorprone excision repair because when synchronized populations of XP variant cells were irradiated at various times prior to S phase to allow different lengths of time for excision repair before DNA replication, the mutant frequency decreased as a function of time for repair until it reached background levels (32). One possible explanation for the UV hypermutability in the variant cells is that their replication fork encounters more photoproducts than does that of normal cells. This would be the case if the slightly lower rate of excision repair sometimes reported for XP variants reflects a significa...

Xeroderma pigmentosum variant (XPV) patients have normal DNA excision repair, yet are predisposed to develop sunlightinduced cancer. They exhibit a 25-fold higher than normal frequency of UV-induced mutations and very unusual kinds (spectrum), mainly transversions. The primary defect in XPV cells is the lack of functional DNA polymerase (Pol) H, the translesion synthesis DNA polymerase that readily inserts adenine nucleotides opposite photoproducts involving thymine. The high frequency and striking difference in kinds of UV-induced mutations in XPV cells strongly suggest that, in the absence of Pol H, an abnormally error-prone polymerase substitutes. In vitro replication studies of Pol I show that it replicates past 5 ¶T-T3 ¶ and 5 ¶T-U3 ¶ cyclobutane pyrimidine dimers, incorporating G or T nucleotides opposite the 3 ¶ nucleotide. To test the hypothesis that Pol I causes the high frequency and abnormal spectrum of UV-induced mutations in XPV cells, we identified an unlimited lifespan XPV cell line expressing two forms of Pol I, whose frequency of UV-induced mutations is twice that of XPV cells expressing one form. We eliminated expression of one form and compared the parental cells and derivatives for the frequency and kinds of UVinduced mutations. All exhibited similar sensitivity to the cytotoxicity of UV (254 nm) , and the kinds of mutations induced were identical, but the frequency of mutations induced in the derivatives was reduced to V50% that of the parent. These data strongly support the hypothesis that in cells lacking Pol H, Pol I is responsible for the high frequency and abnormal spectrum of UV-induced mutations, and ultimately their malignant transformation. [Cancer Res 2007;67(7):3018-26]

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