Thirty percent of the 189 tumors studied to date express DNA polymerase ␤ variants. One of these variants was identified in a prostate carcinoma and is altered from isoleucine to methionine at position 260, within the hydrophobic hinge region of the protein.Another variant was identified in a colon carcinoma and is altered at position 289 from lysine to methionine, within helix N of the protein. We have shown that the types of mutations induced by these cancer-associated variants are different from those induced by the wild-type enzyme. In this study, we show that expression of the I260M and K289M cancer-associated variants in mouse C127 cells results in a transformed phenotype in the great majority of cell clones tested, as assessed by focus formation and anchorageindependent growth. Strikingly, cellular transformation occurs after a variable number of passages in culture but, once established, does not require continuous expression of the polymerase ␤ variant proteins, implying that it has a mutational basis. Because DNA polymerase ␤ functions in base excision repair, our results suggest that mutations that arise during this process can lead to the onset or progression of cancer.base excision repair ͉ DNA repair ͉ mutagenesis S pontaneous DNA damage occurs at a rate of Ϸ10,000 lesions per cell per day, and much of this damage is repaired by the base excision repair (BER) machinery (1, 2). The BER system plays a critical role in maintaining cellular genomic stability. During BER, damaged bases are removed by a DNA glycosylase, followed by incision of the DNA by AP endonuclease (APE) at a position that is usually 5Ј to the lesion, leaving a nick with a 3ЈOH and a 5Ј deoxyribose phosphate (2). DNA polymerase beta (pol ␤) binds to the nick, removes the deoxyribose phosphate with its DRP lyase activity (3), and fills in the single nucleotide gap, using its DNA polymerase activity (4).Fifty-eight of the 189 tumors characterized to date express DNA pol ␤ variant proteins (for review, see ref. 5) (6). Of these, 28 (48%) expressed variants with single amino acid alterations, seven expressed truncated forms of pol ␤, and eight expressed multiple variant forms of pol ␤. These mutations are absent from normal tissue from the same individuals and are not among the common polymorphisms found within the pol ␤ gene (http:͞͞ egp.gs.washington.edu͞data͞polb) (5, 7). In addition, an alternative splice variant of pol ␤ in which exon 11 is deleted was expressed in 15 tumors. This splice variant appears to interfere with BER (8). This exon 11 splice variant has been detected in normal tissue, including normal tissue isolated from 2 of 15 patients with tumors, and its link to cancer etiology remains controversial (9-12). Each of the tumors characterized to date also contain the wild-type (WT) pol ␤ allele.The I260M variant of pol ␤ was identified in a prostate carcinoma (13). Isoleucine 260 is located within a hydrophobic hinge region that appears to function in the movement of the fingers subdomain upon interaction of the polymeras...

Background:Previous small scale studies indicate that DNA polymerase ␤ variants are present in 30% of human tumors. Results: 40% of samples in a large human colorectal tumor collection harbor coding region variants, many of which exhibit altered function. Conclusion: Aberrant activity or fidelity phenotypes exhibited by variants may contribute to tumorigenesis. Significance: Expression of variants in human tumors plays a role in driving carcinogenesis.

DNA polymerase ␤ functions in both base excision repair and meiosis. Errors committed by polymerase ␤ during these processes could result in mutations. Using a complementation system, in which rat DNA polymerase ␤ substitutes for DNA polymerase I of Escherichia coli, we previously isolated a DNA polymerase ␤ mutant in which Tyr-265 was altered to Cys (Y265C). The Y265C mutant is dominant to wild-type DNA polymerase ␤ and possesses an intrinsic mutator activity. We now have expressed the wildtype DNA polymerase and the Y265C mutator mutant in mouse LN12 cells, which have endogenous DNA polymerase ␤ activity. We demonstrate that expression of the Y265C mutator mutant in the LN12 cells results in an 8-fold increase in the spontaneous mutation frequency of cII mutants compared with expression of the wild-type protein. Expression of Y265C results in at least a 40-fold increase in the frequency of deletions of three bases or more and a 7-fold increase in point mutations. Our results suggest that the mutations we observe in vivo result directly from the action of the mutator polymerase. To our knowledge, this is the first demonstration of a mutator phenotype resulting from expression of a DNA polymerase mutator mutant in mammalian cells. This work raises the possibility that variant polymerases may act in a dominant fashion in human cells, leading to genetic instability and carcinogenesis.DNA polymerase ␤ (pol ␤) is a 39-kDa protein with both nucleotidyltransferase and 5Ј-deoxyribose phosphodiesterase activities (1, 2). Evidence has been provided for a role for pol ␤ in both base excision repair and meiosis (3, 4). There is no evidence that pol ␤ functions in replication of the mammalian genome but pol ␤ has been shown to participate in DNA replication in Escherichia coli in the absence of DNA polymerase I (5). However, mice that are completely deficient in pol ␤ die at 10 days postconception, suggesting that pol ␤ is essential for embryonic development (6). The physiological DNA substrate for pol ␤ is believed to be a small gap because it has been shown that pol ␤ is processive on gaps of 6 bases or less and that the activity and fidelity of pol ␤ are highest on a 1-bp gap with a 5Ј phosphate (7,8). Several studies have suggested that pol ␤ is the least accurate mammalian DNA polymerase [for example, see Kunkel (9)], suggesting that gap-filling synthesis by pol ␤ in vivo may result in mutations.To further our understanding of the cellular role of pol ␤, we have identified mutants that are dominant to the wild-type pol ␤ (␤-WT). One of these mutants, termed pol ␤-TR, consists only of amino acid residues 1-170, which comprise a DNA-binding domain of the protein (10). The other mutant, Y265C, is altered from Tyr-265 to Cys (10). This mutant was shown to have an intrinsic mutator activity in three different in vitro assays (11). Using the herpes simplex virus thymidine kinase (HSV-tk) forward mutation assay (12, 13), we found that the Y265C protein commits both base-substitution and frameshift mutations at frequen...