Title: Role of DNA Polymerase η in the UV Mutation Spectrum in Human Cells
Abstract: In humans, inactivation of the DNA polymerase η gene (pol η) results in sunlight sensitivity and causes the cancer-prone xeroderma pigmentosum variant syndrome (XP-V). Cells from XP-V individuals have a reduced capacity to replicate UV-damaged DNA and show hypermutability after UV exposure. Biochemical assays have demonstrated the ability of pol η to bypass cis-syn-cyclobutane thymine dimers, the most common lesion generated in DNA by UV. In most cases, this bypass is error-free. To determine the actual requirement of pol η in vivo, XP-V cells (XP30RO) were complemented by the wild type pol η gene. We have used two pol η-corrected clones to study the in vivo characteristics of mutations produced by DNA polymerases during DNA synthesis of UV-irradiated shuttle vectors transfected into human host cells, which had or had not been exposed previously to UV radiation. The functional complementation of XP-V cells by pol η reduced the mutation frequencies both at CG and TA base pairs and restored UV mutagenesis to a normal level. UV irradiation of host cells prior to transfection strongly increased the mutation frequency in undamaged vectors and, in addition, especially in the pol η-deficient XP30RO cells at 5′-TT sites in UV-irradiated plasmids. These results clearly show the protective role of pol η against UV-induced lesions and the activation by UV of pol η-independent mutagenic processes. In humans, inactivation of the DNA polymerase η gene (pol η) results in sunlight sensitivity and causes the cancer-prone xeroderma pigmentosum variant syndrome (XP-V). Cells from XP-V individuals have a reduced capacity to replicate UV-damaged DNA and show hypermutability after UV exposure. Biochemical assays have demonstrated the ability of pol η to bypass cis-syn-cyclobutane thymine dimers, the most common lesion generated in DNA by UV. In most cases, this bypass is error-free. To determine the actual requirement of pol η in vivo, XP-V cells (XP30RO) were complemented by the wild type pol η gene. We have used two pol η-corrected clones to study the in vivo characteristics of mutations produced by DNA polymerases during DNA synthesis of UV-irradiated shuttle vectors transfected into human host cells, which had or had not been exposed previously to UV radiation. The functional complementation of XP-V cells by pol η reduced the mutation frequencies both at CG and TA base pairs and restored UV mutagenesis to a normal level. UV irradiation of host cells prior to transfection strongly increased the mutation frequency in undamaged vectors and, in addition, especially in the pol η-deficient XP30RO cells at 5′-TT sites in UV-irradiated plasmids. These results clearly show the protective role of pol η against UV-induced lesions and the activation by UV of pol η-independent mutagenic processes. Solar UV induces lesions in genomic DNA. If not repaired by one of the error-free pathways, these lesions can give rise to mutations. In a large percentage of skin tumors, mutations in the p53 tumor suppressor gene are characterized by the "UV mutagenesis signature" namely C → T transitions at pyrimidine-pyrimidine sites and CC → TT tandem mutations (1Brash D.E. Trends Genet. 1997; 13: 410-414Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 2Sarasin A. Mutat. Res. 1999; 428: 5-10Crossref PubMed Scopus (176) Google Scholar). The precise mechanism by which UV-induced damage results in mutations remains unclear. The frequency and the nature of mutation depends on the types of initial DNA damage, their potential to miscode during DNA replication, and the probability that specific enzymes act on a given type of damage. The mutational specificity of UV light correlates with the formation in DNA of the two predominant UV-induced lesions, the cis-syn cyclobutane pyrimidine dimer (CPD) 1The abbreviations used are: CPD, cis-syn cyclobutane pyrimidine dimer; MER, mutation error rate; XP-V, xeroderma pigmentosum variant; BrdUrd, bromodeoxyuridine; pol η, polymerase η; 6-4PP, pyrimidine-6/4-pyrimidone; TLS, translesion synthesis.1The abbreviations used are: CPD, cis-syn cyclobutane pyrimidine dimer; MER, mutation error rate; XP-V, xeroderma pigmentosum variant; BrdUrd, bromodeoxyuridine; pol η, polymerase η; 6-4PP, pyrimidine-6/4-pyrimidone; TLS, translesion synthesis. and the pyrimidine-6/4-pyrimidone (6-4PP) photoproducts (3Sage E. Photochem. Photobiol. 1993; 57: 163-174Crossref PubMed Scopus (213) Google Scholar, 4Tornaletti S. Pfeifer G.P. Bioessays. 1996; 18: 221-228Crossref PubMed Scopus (129) Google Scholar). In naked DNA, a similar distribution of the main UV-induced photoproducts was obtained with either UVC (254 nm) or UVB (280–320 nm). In both cases, the T = T CPD is the most abundant photoproduct. The T = C CPD and the T(6-4)C damage are produced in similar yields, whereas the level of the T(6-4)T lesion and photoproducts at CT and CC sites remains much lower (5Douki T. Cadet J. Biochemistry. 2001; 40: 2495-2501Crossref PubMed Scopus (257) Google Scholar). These DNA lesions are normally repaired by the nucleotide excision repair system (6de Laat W.L. Jaspers N.G. Hoeijmakers J.H. Genes Dev. 1999; 13: 768-785Crossref PubMed Scopus (911) Google Scholar). The 6-4PPs are rapidly and efficiently removed by nucleotide excision repair within a few hours after cell irradiation, whereas the CPDs are repaired rather slowly and incompletely (7Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar). Although individual unrepaired 6-4PPs are more mutagenic than CPDs (8Gentil A. Le Page F. Margot A. Lawrence C.W. Borden A. Sarasin A. Nucleic Acids Res. 1996; 24: 1837-1840Crossref PubMed Scopus (72) Google Scholar, 9Lawrence C.W. Gibbs P.E. Borden A. Horsfall M.J. Kilbey B.J. Mutat. Res. 1993; 299: 157-163Crossref PubMed Scopus (57) Google Scholar), CPDs are thought to be responsible for the majority of the premutagenic lesions in mammalian cells (10Kamiya H. Iwai S. Kasai H. Nucleic Acids Res. 1998; 26: 2611-2617Crossref PubMed Scopus (65) Google Scholar, 11Marionnet C. Armier J. Sarasin A. Stary A. Cancer Res. 1998; 58: 102-108PubMed Google Scholar, 12You Y.H. Lee D.H. Yoon J.H. Nakajima S. Yasui A. Pfeifer G.P. J. Biol. Chem. 2001; 276: 44688-44694Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), probably because the CPDs are more frequent and repaired much less efficiently than the 6-4PP lesions. Although T = T CPDs are relatively more frequent than CPD containing cytosine, UV-induced mutations occur predominantly at 5′-TC-3′ and 5′-CC-3′ dipyrimidine sites, particularly in vivo in key genes found mutated in skin cancers. In most cases they are C → T transitions, arising from the misincorporation of an adenine opposite the 3′-C of 5′-TC-3′ and 5′-CC-3′ sites. The weak contribution of T = T dimers to the UV mutation spectrum in vivo could be explained by a specific and accurate DNA damage tolerance mechanism. UV-induced DNA damage presents a strong block for the replication machinery, probably because of the inability of replicative DNA polymerases to deal with DNA distortions. Specialized DNA polymerases are temporarily required to perform translesion synthesis (TLS) (13Friedberg E.C. Wagner R. Radman M. Science. 2002; 296: 1627-1630Crossref PubMed Scopus (389) Google Scholar, 14Lehmann A.R. Gene (Amst.). 2000; 253: 1-12Crossref PubMed Scopus (45) Google Scholar, 15Wang Z. Mutat. Res. 2001; 486: 59-70Crossref PubMed Scopus (85) Google Scholar, 16Woodgate R. Genes Dev. 1999; 13: 2191-2195Crossref PubMed Scopus (234) Google Scholar). In bacteria, when DNA damage blocks the normal replication process, an "SOS" response is activated, and more than 20 genes are induced (17Goodman M.F. Annu. Rev. Biochem. 2002; 71: 17-50Crossref PubMed Scopus (620) Google Scholar, 18Sutton M.D. Walker G.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8342-8349Crossref PubMed Scopus (153) Google Scholar). Among these genes, two DNA polymerases (DNA polymerases IV and V) are able to continue to synthesize DNA for a few bases across the damaged site and put an incorrect base opposite the lesion, producing a mutation. In yeast, three genes (REV1, REV3, and REV7) are required for most DNA damage-induced mutagenesis. Rev3 and Rev7 code for a specific mutagenic DNA polymerase activity (pol ζ) required for translesion synthesis and the Rev1 gene product is a dCMP transferase (19Nelson J.R. Lawrence C.W. Hinkle D.C. Science. 1996; 272: 1646-1649Crossref PubMed Scopus (592) Google Scholar). In parallel to this error-prone activity, yeast cells have another specialized DNA polymerase, DNA polymerase η encoded by the RAD30 gene (pol η), that can perform translesion synthesis in a relatively error-free way (20Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (690) Google Scholar). Most DNA polymerases associated with TLS belong to the Y family of DNA polymerases that have in common a highly distributive mode of DNA synthesis and copy undamaged DNA templates with low fidelity (21Friedberg E.C. Fischhaber P.L. Kisker C. Cell. 2001; 107: 9-12Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 22Matsuda T. Bebenek K. Masutani C. Hanaoka F. Kunkel T.A. 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Nature. 1999; 399: 700-704Crossref PubMed Scopus (1134) Google Scholar), suggesting that it is able to carry out error-free TLS past T = T CPDs in vivo. The relevance of human pol η to mutagenesis in vivo is demonstrated by the existence of patients mutated in the human pol η gene (27Johnson R.E. Kondratick C.M. Prakash S. Prakash L. Science. 1999; 285: 263-265Crossref PubMed Scopus (665) Google Scholar, 28Masutani C. Araki M. Yamada A. Kusumoto R. Nogimori T. Maekawa T. Iwai S. Hanaoka F. EMBO J. 1999; 18: 3491-3501Crossref PubMed Scopus (384) Google Scholar). These patients are affected by the variant form of xeroderma pigmentosum (XP-V), a rare, autosomal, recessive human genetic syndrome with sun hypersensitivity associated with numerous skin abnormalities and a high level of early and multiple skin cancers on sun-exposed sites in the body (29Berneburg M. Lehmann A.R. Adv. Genet. 2001; 43: 71-102Crossref PubMed Google Scholar, 30Kraemer K.H. Lee M.M. Scotto J. Arch. Dermatol. 1987; 123: 241-250Crossref PubMed Scopus (948) Google Scholar). The phenotype of XP-V cells includes UV hypersensitivity, especially in the presence of caffeine (31Arlett C.F. Harcourt S.A. Broughton B.C. Mutat. Res. 1975; 33: 341-346Crossref PubMed Scopus (122) Google Scholar), and UV-hypermutability that is consistent with the high cancer proneness in XP-V patients (32Maher V.M. Ouellette L.M. Curren R.D. McCormick J.J. Nature. 1976; 261: 593-595Crossref PubMed Scopus (266) Google Scholar, 33McGregor W.G. Wei D. Maher V.M. McCormick J.J. Mol. Cell. Biol. 1999; 19: 147-154Crossref PubMed Scopus (68) Google Scholar, 34Wang Y.C. Maher V.M. McCormick J.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7810-7814Crossref PubMed Scopus (64) Google Scholar, 35Wang Y.C. Maher V.M. Mitchell D.L. McCormick J.J. Mol. Cell. Biol. 1993; 13: 4276-4283Crossref PubMed Scopus (141) Google Scholar, 36Waters H.L. Seetharam S. Seidman M.M. Kraemer K.H. J. Invest. Dermatol. 1993; 101: 744-748Abstract Full Text PDF PubMed Google Scholar). XP-V cells are proficient in nucleotide excision repair but are impaired in lesion bypass associated with DNA replication on damaged templates (37Lehmann A.R. Kirk-Bell S. Arlett C.F. Paterson M.C. Lohman P.H. de Weerd-Kastelein E.A. Bootsma D. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 219-223Crossref PubMed Scopus (523) Google Scholar). Cell-free extracts of XP-V patients are unable to perform bypass through a T = T dimer (38Cordeiro-Stone M. Zaritskaya L.S. Price L.K. Kaufmann W.K. J. Biol. Chem. 1997; 272: 13945-13954Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 39Ensch-Simon I. Burgers P.M. Taylor J.S. Biochemistry. 1998; 37: 8218-8226Crossref PubMed Scopus (33) Google Scholar, 40Svoboda D.L. Briley L.P. Vos J.M. Cancer Res. 1998; 58: 2445-2448PubMed Google Scholar), a T (6-4) C photoproduct (41Yao J. Dixon K. Carty M.P. Environ. Mol. Mutagen. 2001; 38: 19-29Crossref PubMed Scopus (15) Google Scholar), or other bulky DNA damage (42Cordonnier A.M. Lehmann A.R. Fuchs R.P. Mol. Cell. Biol. 1999; 19: 2206-2211Crossref PubMed Scopus (69) Google Scholar). Restoration of efficient DNA synthesis past a T = T dimer by the addition of pol η protein in cell-free extracts demonstrated the essential role of pol η in replication of this photoproduct in vitro (26Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. Nature. 1999; 399: 700-704Crossref PubMed Scopus (1134) Google Scholar). Complementation of UV + caffeine hypersensitivity after transfection of the human pol η cDNA into XP-V cells definitively assigned pol η as the gene responsible for the genetic defect in XP-V patients (43Kannouche P. Broughton B.C. Volker M. Hanaoka F. Mullenders L.H. Lehmann A.R. Genes Dev. 2001; 15: 158-172Crossref PubMed Scopus (243) Google Scholar, 44Thakur M. Wernick M. Collins C. Limoli C.L. Crowley E. Cleaver J.E. Genes Chromosomes Cancer. 2001; 32: 222-235Crossref PubMed Scopus (44) Google Scholar, 45Yamada A. Masutani C. Iwai S. Hanaoka F. Nucleic Acids Res. 2000; 28: 2473-2480Crossref PubMed Scopus (76) Google Scholar). Most mutations in the pol η gene in XP-V patients are heavily biased toward the N-terminal region and encode a pol η protein with either mis-sense mutations or severe truncations that abolish both DNA polymerase and bypass activities (46Broughton B.C. Cordonnier A. Kleijer W.J. Jaspers N.G. Fawcett H. Raams A. Garritsen V.H. Stary A. Avril M.F. Boudsocq F. Masutani C. Hanaoka F. Fuchs R.P. Sarasin A. Lehmann A.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 815-820Crossref PubMed Scopus (141) Google Scholar). Complementation of the defect in XP-V cells required not only the N-terminal catalytic domain of pol η but also the C-terminal 120 amino acids containing a bipartite nuclear localization signal and a sequence needed for the relocalization into replication foci. Indeed, in normal cells, pol η is distributed most uniformly in the nucleoplasm in most cells, but following UV irradiation, it accumulates with proliferating cell nuclear antigen in intranuclear foci, which represent forks stalled at sites of unrepaired DNA damage (43Kannouche P. Broughton B.C. Volker M. Hanaoka F. Mullenders L.H. Lehmann A.R. Genes Dev. 2001; 15: 158-172Crossref PubMed Scopus (243) Google Scholar). To evaluate the contribution of pol η to UV mutagenesis in vivo, we compared the UV-induced mutation spectra produced in the pol η-deficient XP-V cell line (XP30RO) and in stable pol η-complemented clones derived from these cells. We assessed by DNA sequencing the error rates and the types of mutations generated by replication of UVC-irradiated SV40-based shuttle vectors in unirradiated or UVC-irradiated human host cells. Because replication of UV-damaged plasmids requires the human cellular replication machinery, the level and the kind of mutagenic TLS are dependent on the presence of pol η in host human cells and can be modulated by the localization of pol η in replication foci after UV radiation. Our data demonstrate that the expression of pol η in XP variant cells gives rise to a strong decrease in the number of errors generated during DNA synthesis past UV photoproducts, especially at T = T lesions in UV-irradiated host cells but also at C sites. UV irradiation of host cells prior to replicating plasmids induces an error-prone mode of replication that is pol η-independent on undamaged plasmids, but pol η-dependent on damaged plasmids. Cell Lines and Culture Conditions—All experiments were performed using SV40-transformed MRC5V1 (normal) and XP30RO human fibroblasts (47Volpe J.P. Cleaver J.E. Mutat. Res. 1995; 337: 111-117Crossref PubMed Scopus (14) Google Scholar). The XP30RO cell line has a homozygous deletion near the 5′-extremity of the pol η gene leading to a severe truncation of the pol η protein (26Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. Nature. 1999; 399: 700-704Crossref PubMed Scopus (1134) Google Scholar, 46Broughton B.C. Cordonnier A. Kleijer W.J. Jaspers N.G. Fawcett H. Raams A. Garritsen V.H. Stary A. Avril M.F. Boudsocq F. Masutani C. Hanaoka F. Fuchs R.P. Sarasin A. Lehmann A.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 815-820Crossref PubMed Scopus (141) Google Scholar). Isolation of complemented cells by the wild type pol η gene has been described previously (43Kannouche P. Broughton B.C. Volker M. Hanaoka F. Mullenders L.H. Lehmann A.R. Genes Dev. 2001; 15: 158-172Crossref PubMed Scopus (243) Google Scholar). Stable transfectants isolated from transfection of pCDNA.3zeo-pol η plasmid or vector with no insert were grown in modified Eagle's medium supplemented with 10% fetal bovine serum, fungizone (2.5 μg/ml), antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin), and 100 μg/ml Zeocin (Invitrogen). MRC5V1 cl1 and XP30RO cl4 harboring the pCDNA.3zeo (Invitrogen) plasmid without the pol η insert were utilized as control cell lines and are referred to MRC5 and XP30RO cells. UV Survival Assay—Cells were plated at 100–1000 cells per 100-mm dishes and incubated for 24 h. They were then rinsed with phosphate-buffered saline and irradiated with 254 nm UV light using a germicidal lamp at a fluence rate of 0.24 J/m2/s. After 15 days of incubation in culture medium containing 75 μg/ml caffeine (Sigma), the colonies were fixed with methanol and stained with Giemsa, and colonies with >50 cells were counted. Three dishes were utilized for each dose. Cell Cycle Analysis—Twenty four hours after exposure to 0 or 7 J/m2 of UVC and caffeine (75 μg/ml) added to the medium, cells were pulsed with BrdUrd for 1 h, harvested, and stained for replicative DNA synthesis with a fluorescein isothiocyanate-conjugated anti-BrdUrd antibody and for DNA content with propidium iodide. Mutagenesis Assay—The shuttle vector SV40-based pR2 carries the 300-bp bacterial lacZ′ gene and bacterial promoter as mutagenesis target, the kanamycin resistance gene, and the SV40 and bacterial replication origins (48Marionnet C. Benoit A. Benhamou S. Sarasin A. Stary A. J. Mol. Biol. 1995; 252: 550-562Crossref PubMed Scopus (34) Google Scholar). The p205-KMT11 plasmid carries the SV40 T-antigen gene required for replication of the pR2 plasmid (49Stary A. Menck C.F. Sarasin A. Mutat. Res. 1992; 272: 101-110Crossref PubMed Scopus (30) Google Scholar). Both vectors were transfected together into the host cells. The RecA-deficient Escherichia coli DH5αMCR (Invitrogen) was used for screening of lacZ′ mutants among plasmid progeny. Irradiation of plasmid DNA was performed with 254 nm UV light using a germicidal lamp at a fluence rate of 30 J/m2/s. Cells were irradiated at 7 J/m2 with 254 nm UV light using a germicidal lamp at a fluence rate of 0.24 J/m2/s. Cells were seeded at a density of 5 × 105 per 100-mm dish and incubated for 24 h. Undamaged DNA (10 μg of plasmid DNA) or UV-exposed pR2 vectors (10 μg of plasmid DNA) were then cotransfected with the 5 μg of unirradiated p205-KMT11 plasmid into unirradiated or irradiated cells at 7 J/m2 just before DNA transfection by the polyethyleneimine precipitation method (50Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5526) Google Scholar). Cells were incubated for 4 days and then collected by trypsinization for extrachromosomal DNA extraction. At least three independent cell transfections were performed for each dose and for each cell line. Plasmid DNA isolated from cells was purified by a small scale alkaline lysis method (51Stary A. Sarasin A. J. Gen. Virol. 1992; 73: 1679-1685Crossref PubMed Scopus (45) Google Scholar) and treated with DpnI restriction endonuclease to remove any unreplicated pR2. Rescued plasmid DNA was shuttled to competent E. coli DH5αMCR for selection of mutant vectors, and transformed colonies were plated on selective medium (48Marionnet C. Benoit A. Benhamou S. Sarasin A. Stary A. J. Mol. Biol. 1995; 252: 550-562Crossref PubMed Scopus (34) Google Scholar). The α-complementation between the lacZ′ gene carried by the pR2 vector and the truncated lacZ gene of DH5α bacteria gives rise to blue (wild type) or white/light blue (mutant) colonies. Selected colonies were then isolated and restreaked on the same medium to confirm the mutant phenotype. Plasmid DNA was prepared by small scale alkaline lysis, and sequence analysis at the lacZ′ locus was performed with Big Dye terminator on an ABI Prism 377 DNA sequencer. Mutant frequency corresponds to the number of colonies containing mutated plasmid at the lacZ′ locus determined after DNA sequencing divided by the total number of bacterial colonies. Mutation error rate (MER) corresponds to the average of the frequency of each independent mutation calculated as the ratio of each independent mutation divided by the total number of bacterial colonies rescued from the cell transfection experiment in which the mutation was isolated. Identical mutations that occurred more than once among sequences analyzed from the same cell transfection were excluded, thus ensuring that each mutation is the result of one event of bypass process and not of several rounds of replication of one mutated plasmid during the 4-day postincubation after DNA transfection. Although this will lead to a slight underestimation of the mutation frequency at hotspot sites, the number of individual transfections is sufficient enough to ensure that no major hot spots are missed. Statistical Analysis—The significance of differences of percentages was assessed using χ2 or Fisher's exact test when appropriate, and the significance of differences of mutant and mutation error rates was assessed with the Student's t test. Mutation hotspot analysis was carried out according to the Poisson law at a probability of less than 1%. The hypergeometric test was used to check the significant differences in the distribution of mutations between spectra (52Adams W.T. Skopek T.R. J. Mol. Biol. 1987; 194: 391-396Crossref PubMed Scopus (261) Google Scholar, 53Cariello N.F. Cui L. Beroud C. Soussi T. Cancer Res. 1994; 54: 4454-4460PubMed Google Scholar). Complementation of UV Sensitivity of XP-V Cells by the Human pol η cDNA—Stable clones expressing the pol η protein were isolated after transfection of pCDNA-pol η plasmid into pol η-deficient XP30RO cells as described previously (43Kannouche P. Broughton B.C. Volker M. Hanaoka F. Mullenders L.H. Lehmann A.R. Genes Dev. 2001; 15: 158-172Crossref PubMed Scopus (243) Google Scholar). Among the stable XP30RO transfectants, we characterized two clones, XP30RO/pol η cl5 and XP30RO/pol η cl6 cells, for their resistance to UV irradiation in the presence of caffeine, which specifically enhances UV sensitivity of XP-V cells, thereby allowing a clear discrimination between XP-V and normal cells (31Arlett C.F. Harcourt S.A. Broughton B.C. Mutat. Res. 1975; 33: 341-346Crossref PubMed Scopus (122) Google Scholar). The expression of wild type pol η protein, determined by Western blot analysis (Fig. 1) in the two complemented clones, is similar in XP30RO/pol η cl5 and normal MRC5 cells and slightly higher in XP30RO/pol η cl6 cells. As expected no pol η was detected in the parental XP30RO cells. The UV sensitivity of XP30RO/pol η cl5 and XP30RO/pol η cl6 clones was monitored by cell cycle analysis and cell survival after UVC irradiation and was compared with that of XP30RO and normal MRC5 fibroblasts, both transfected by vector without insert. The distribution of cells in the G1, S, and G2/M phases of the cell cycle 24 h after UV + caffeine treatment are shown in Fig. 2A, and a quantitative assessment of cell cycle distribution is illustrated in Fig. 2B. In the absence of UV, the number of BrdUrd-labeled cells in S phase was similar in all cell lines (18–25%). UV irradiation and the addition of caffeine did not substantially change the proportion of BrdUrd-labeled XP30RO/pol η cl5 (28%), XP30RO/pol η cl6 (24%), or MRC5 cells (34%) in S phase 24 h after irradiation. In contrast, only a very small number of XP30RO cells (3%) incorporated BrdUrd. Examination of the distribution of DNA content in these cells (Fig. 2A, lower row, 2nd panel) shows that many of them were blocked in S phase by the UV + caffeine treatment. Thus, pol η is able to prevent the prolonged delay in S phase after UV irradiation typical of XP-V cells. The differential effect of UVC on XP30RO/pol η cl5, XP30RO/pol η cl6, and XP30RO cells was also demonstrated by examining cell survival after UVC exposure and caffeine post-treatment with a colony forming ability assay (Fig. 2C). The survival curves of XP30RO/pol η cl5 and XP30RO/pol η cl6 cells were indistinguishable from that of normal MRC5 cells, whereas XP30RO cells were extremely sensitive to UV irradiation in the presence of caffeine (3% at 4 J/m2), as expected for XP-V cells. A similar result was obtained when viability was assessed by the ability of the cells to incorporate [3H]thymidine 2–4 days after exposure to different doses of UV in the presence of caffeine (46Broughton B.C. Cordonnier A. Kleijer W.J. Jaspers N.G. Fawcett H. Raams A. Garritsen V.H. Stary A. Avril M.F. Boudsocq F. Masutani C. Hanaoka F. Fuchs R.P. Sarasin A. Lehmann A.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 815-820Crossref PubMed Scopus (141) Google Scholar) (data not shown). The expression of pol η in XP30RO/pol η cl5 and XP30RO/pol η cl6 cells therefore results in the correction of UV sensitivity to normal levels. The functional complementation of XP30RO/pol η cl5 and XP30RO/pol η cl6 cells compared with their parental counterpart XP30RO cells deficient in the expression of pol η gave us the opportunity to examine the outcome of the expression of pol η on UV mutagenesis in vivo. Plasmid Mutagenesis—The SV40-based shuttle vector pR2 (48Marionnet C. Benoit A. Benhamou S. Sarasin A. Stary A. J. Mol. Biol. 1995; 252: 550-562Crossref PubMed Scopus (34) Google Scholar) was irradiated with different UV doses (500, 1000, or 2000 J/m2) and immediately transfected into XP30RO/pol η cl5, XP30RO/pol η cl6, XP30RO, and MRC5 cells. In some experiments the recipient cells were pre-irradiated with a UV dose of 7 J/m2. Cells were further incubated for 4 days to allow DNA replication and mutation fixation to take place. The plasmid DNAs were recovered from human cells and shuttled into E. coli for mutant plasmid screening at the nonessential lacZ′ locus. Phenotypic selection of lacZ′ mutants followed by DNA sequencing enabled us to determine the mutant error rates and the sequence alterations. Unirradiated plasmid DNA replication in unirradiated host cells (Fig. 3A) led to similar background mutant frequencies in the parental XP30RO cells (39 × 10–5) and in the corrected XP30RO/pol η cl5 and XP30RO/pol η cl6 cells (24 × 10–5 and 17 × 10–5 respectively). Mutant frequencies after replication of UV-irradiated shuttle vectors were increased significantly in all cells relative to the background levels. However, when UV-damaged plasmids replicated in XP30RO cells, there was a 5.1-fold increase in mutant frequencies from 39 × 10–5 to 200 × 10–5 (Student's t test; p < 0.001), reflecting the cellular hypermutability of XP variant cells. The mutant error rates in XP30RO cells at all doses of plasmid irradiation were much higher than those in the XP30RO/pol η cl5, XP30RO/pol η cl6, and MRC5 cells (p < 0.001). The transfected wild type pol η gene thus complements the high UV-induced mutant frequency characteristic of XP-V cells. After introduction of plasmid DNA into UV-exposed cells (7 J/m2) (Fig. 3B), there was an increase of the background frequencies in all cells, changing from 39 × 10–5 to 213 × 10–5 for XP30RO cells (5.5 times), from 24 × 10–5 to 165 × 10–5 for XP30RO/pol η cl5 cells (6.9 times), and from 17 × 10–5 to 87 × 10–5 for XP30RO/pol η cl6 cells (5.1 times). Since the global enhancement of the mutant error rate affected both pol η-deficient and -proficient cells, the cellular process involved in this increase is independent of the cellular status of pol η. The UV-induced mutant frequency in XP30RO cells (787 × 10–5 and 565 × 10–5 at 1000 and 2000 J/m2 doses of plasmid irradiation), however, remained substantially higher than the background mutant error rate (p < 0.02). In contrast, the mutant frequencies in XP30RO/pol η cl5 and XP30RO/pol η cl6 cells were not significantly different from the background frequencies. These data show that the expression of pol η in XP variant cells gives rise to a strong decrease in errors generated during replication of UV lesions. Sequence Analysis—DNA sequence analysis of more than 300 independent undamaged and UV-induced mutants in the lacZ′ loc