Previous investigations have shown that Ϸ35% of the 90 tumors analyzed to date contain mutations within the DNA polymerase ␤ (pol ␤) gene. The existence of pol ␤ mutations in a substantial fraction of human tumors studied suggests a link between DNA pol ␤ and cancer. A DNA pol ␤ variant, in which Lys-289 has been altered to Met, was identified previously in a colorectal carcinoma. The K289M protein was expressed in mouse L cells containing the cII mutational target. The DNA was packaged and used to infect bacterial cells to obtain the spontaneous mutation frequency. We found that expression of K289M in the mouse cells resulted in a 2.5-fold increase in the mutation frequency. What was most interesting was that expression of K289M in these cells resulted in a 16-fold increase in the frequency of C to G or G to C base substitutions at a specific site within the cII target. By using this cII target sequence, kinetic analysis of the purified K289M protein revealed that it was able to misincorporate dCTP opposite template C and dGTP opposite template G with significantly higher efficiency than the wild-type pol ␤ protein. We provide evidence that misincorporation of nucleotides by K289M results from altered positioning of the DNA within the active site of the enzyme. Our data are consistent with the interpretation that misincorporation of nucleotides resulting from altered DNA positioning by the K289M protein has the potential to result in tumorigenesis or neoplastic progression.M utations in the gene encoding DNA polymerase ␤ (pol ␤) have been identified in human colorectal, prostate, lung, and breast carcinomas and mouse lymphomas (1-5). Thus far, only 90 tumors have been analyzed for mutations within the pol ␤ coding sequence, and mutations are present in 35% of these tumors. The pol ␤ tumor-associated mutations are found only in the tumor, and not in normal tissue from the same patient, implying that they represent sporadic mutations underlying neoplastic disease. Furthermore, the mutations identified in these tumors are not present in the pol ␤ gene of 124 normal individuals (6). Also of interest is that pol ␤ is located within the proximal region of the short arm of chromosome 8 (p12-p11), a region that is frequently lost in a variety of human tumors, including colorectal and prostate carcinomas (7). These studies suggest a link between mutations within the pol ␤ gene and carcinogenesis. Another piece of evidence that is consistent with a role for pol ␤ in cancer is its interaction with the tumor suppressor protein p53 (8, 9). The p53 protein stabilizes pol ␤ at an abasic site. An alteration of the p53-pol ␤ interaction could result in less efficient DNA repair, which may contribute to the development of neoplastic disease. Most interestingly, a pol ␤ mutant with an 87-bp deletion, which has been found in primary colorectal, lung, and breast adenocarcinomas (3,5,10), is dominant to the WT enzyme and disrupts its base excision repair (BER) activity if expressed in human cell lines (11,12). It is quite possible t...

DNA polymerase beta offers an attractive system to study the biochemical mechanism of polymerase-dependent mutagenesis. Variants of DNA polymerase beta, Y265F and Y265W, were analyzed for misincorporation efficiency and mispair extension ability, relative to wild-type DNA polymerase beta. Our data show that the fidelity of the mutant polymerases is similar to wild-type enzyme on a one-nucleotide gapped DNA substrate. In contrast, with a six-nucleotide gapped DNA, the mutant proteins are slightly more accurate than the wild-type enzyme. The mutagenic potential of Y265F and Y265W is more pronounced when encountering a mispaired DNA substrate. Here, both variants can extend a G:G mispair quite efficiently, and Y265F can also extend a T:G mispair. The kinetic basis of the increased mispair extension efficiency is due to an improved ability to bind to the incoming nucleotide. Y265W extends the G:G mispair even with an incorrect nucleotide substrate. Overall, our results demonstrate that the Y265 hinge residue is important for stabilizing the architecture of the nucleotide binding pocket of DNA polymerase beta, and that alterations of this residue can have significant impacts upon the fidelity of DNA synthesis.

DNA polymerase ␤ (pol ␤) is an ideal system for studying the role of its different amino acid residues in the fidelity of DNA synthesis. In this study, the T79S variant of pol ␤ was identified using an in vivo genetic screen. T79S is located in the N-terminal 8-kDa domain of pol ␤ and has no contact with either the DNA template or the incoming dNTP substrate. The T79S protein produced 8-fold more multiple mutations in the herpes simplex virus type 1-thymidine kinase assay than wild-type pol ␤. Surprisingly, T79S is a misincorporation mutator only when using a 3-recessed primer-template. In the presence of a single nucleotide-gapped DNA substrate, T79S displays an antimutator phenotype when catalyzing DNA synthesis opposite template C and has similar fidelity as wild type opposite templates A, G, or T. Threonine 79 is located directly between two helix-hairpinhelix motifs located within the 8-kDa and thumb domains of pol ␤. As the pol ␤ enzyme closes into its active form, the helix-hairpin-helix motifs appear to assist in the production and stabilization of a 90 o bend of the DNA. The function of the bent DNA is to present the templating base to the incoming nucleotide substrate. We propose that Thr-79 is part of a hydrogen bonding network within the helix-hairpin-helix motifs that is important for positioning the DNA within the active site. We suggest that alteration of Thr-79 to Ser disrupts this hydrogen bonding network and results in an enzyme that is unable to bend the DNA into the proper geometry for accurate DNA synthesis.1 has quickly become one of the best studied polymerases because the gene for the enzyme was cloned (1, 2). The availability of multiple crystal structures of human and rat pol ␤, including those of the enzyme complexed with both of its substrates and the metal cofactor, has aided the investigation of the structure-function relationships of this enzyme (3-8).pol ␤ is a 39-kDa protein with both nucleotidyltransferase and 5Ј-deoxyribose phosphodiesterase activities (9, 10). Evidence has been provided for a role for pol ␤ in both base excision repair and meiosis (11-13). 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 (14). Mice that are completely deficient in pol ␤ die at 18 days post-conception due to massive apoptosis of post-mitotic neurons, suggesting that pol ␤ is essential for embryonic development (15, 16). 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 (17, 18).DNA polymerase ␤ has a modular organization with an 8-kDa N-terminal domain connected to the 31-kDa C-terminal domain by a protease-hypersensitive hinge region. The N-terminal 8-kDa domain was originally characterized as a singlestranded DNA binding domain (19,20). Subsequently, it was foun...

We have studied the effects of ARS addition and deletion on the maintenance of a 61-kb ring derivative of chromosome III in a minichromosome maintenance mutant of yeast carrying the mcm2-1 mutation. When this ring chromosome, CIIIR, had either of its two strong origins deleted, the resultant chromosome showed a much greater instability in the mutant as compared to that of the wild-type strain. Integration of more ARSs improved the maintenance of CIIIR in the mutant but not in the wild-type strain. Increase in the size of CIIIR, without any ARS addition, did not improve the stability in either strain. A spontaneous revertant for improved growth at 35 degrees C also co-reverted for minichromosome and CIIIR maintenance. The results suggest that ARS malfunctioning leads to minichromosome and chromosome loss from mutant cells, affecting their growth at higher temperatures.