In Saccharomyces cerevisiae, most mutations induced by a wide range of mutagens arise during translesion replication employing the REV1 gene product and DNA polymerase . As part of an effort to investigate mammalian mutagenic mechanisms, we have identified cDNA clones of the human homologs of the yeast REV genes and examined their function in UV mutagenesis. Previously, we described the isolation of a human homolog of yeast REV3, the catalytic subunit of pol , and here report the identification and sequence of a human homolog of yeast REV1. This gene was isolated by identifying an expressed sequence tag encoding a peptide with similarity to the C terminus of yeast Rev1p, followed by sequencing of the clone and retrieval of the remaining cDNA by 5 rapid amplification of cDNA ends. The human gene encodes an expected protein of 1,251 residues, compared with 985 residues in the yeast protein. The proteins share two amino-terminal regions of Ϸ100 residues with 41% and 20% identity, a region of Ϸ320 residues with 31% identity, and a central motif in which 11 of 13 residues are identical. Human cells expressing high levels of an hREV1 antisense RNA grew normally, and were not more sensitive to the cytotoxic effect of 254 nm UV radiation than cells lacking antisense RNA. However, the frequencies of 6-thioguanine resistance mutants induced by UV in the cells expressing antisense hREV1 RNA were significantly lower than in the control (P ؍ 0.01), suggesting that the human gene has a function similar to that of the yeast homolog.
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]
Translesion synthesis (TLS) refers to mechanisms by which specialized DNA polymerases incorporate nucleotides opposite fork-blocking lesions and extend replication until standard replicative polymerases take over. The first eukaryotic TLS polymerase discovered, S. cerevisiae Polzeta, consists of catalytic subunit Rev3 and non-catalytic subunit Rev7. Human homologs of these two proteins have been identified. Studies by Lawrence, Maher, and colleagues comparing UV((254nm))-irradiated human fibroblast cell strains expressing high levels of hRev3 antisense to their normal parental strains demonstrated that there was no difference in cell survival, but that the frequency of UV-induced mutations in the derivative strains was 10-fold lower than that of the parental strains, indicating that hRev3 plays a critical role in such mutagenesis. To examine the role of hRev7 in TLS, we generated human fibroblasts expressing hRev7 siRNA, identified two derivative cell strains with significantly reduced levels of hRev7, and compared them to their parental strain and a vector control for cell survival, induction of mutations, and ability to traverse the cell cycle following exposure to UV radiation. Cells with reduced hRev7 were approximately 2-times more sensitive to UV-induced cytotoxicity than the controls, indicating that unlike hRev3, hRev7 plays a protective role for cells exposed to UV radiation. When these cell strains were assayed for the frequency of mutations induced by UV in their HPRT gene, cell stains with reduced hRev7 were 5-times less sensitive to UV-induced mutagenesis than control strains. In addition, when these four strains were synchronized at the G1/S border, released from the block, UV-irradiated, and allowed to traverse the cell cycle, the rate of progression through S-phase of the cell strains with reduced hRev7 was significantly slower than that of the control strains. These data strongly support the hypothesis that hRev7 is required for TLS past UV-photoproducts, and together with hRev3, comprise hPolzeta.
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