A DNA polymerase β mutant from colon cancer cells induces mutations

Abstract

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...

Is Base Excision Repair a Tumor Suppressor Mechanism?

“…[15][16][17] In support of this idea we demonstrated that both I260M and K289M are sequence-specific mutator mutants. 13,12 Because Pol β functions in BER, our results suggest that K289M and I260M are deficient in discriminating between the correct and incorrect dNTP as they fill the gap after excision of the damaged base. If mistakes are introduced into key growth control genes during gap filling, they could result in mutations that lead to cancer.…”

“…[11][12][13] Specifically, expression of either the K289M colon or I260M prostate cancer Pol β variant in mouse fibroblasts, in the presence of endogenous wild-type Pol β, results in cellular transformation as assessed by focus formation and anchorage independent growth. [11][12][13] A mutagenic basis for the transforming activity of the Pol β variants is suggested by the findings that different clones of cells expressing these polymerases were not immediately transformed but acquired the transformed phenotype after a variable number of passages and that the transformed phenotype persisted even after the expression of the Pol β variant was extinguished. The latter finding indicates that the Pol β variants induce transformation by a "hit-and-run" mechanism in which the continued expression of the initiating event is not required to maintain the transformed state.…”

Expression of DNA polymerase β cancer-associated variants in mouse cells results in cellular transformation

“…I260M induces expansions of TC repeats within a run of TCs, frameshift mutations within dipyrimidine sequences, and base substitutions within sequence contexts in which WT pol ␤ does not induce mutations (S.D., K. Eckert, and J.B.S., unpublished data). The K289M variant induces transversions within interrupted runs of like residues at frequencies that are much greater than those of the WT protein (24). The molecular basis of mutation induction by the I260M and K289M proteins appears to be altered positioning of the DNA within the active site of each of these enzymes as compared with WT pol ␤.…”

Human POLB Gene Is Mutated in High Percentage of Colorectal Tumors

“…We have shown that K289M variant protein induces cellular transformation in immortalized mouse epithelial cells by increasing the mutation frequency of the cells (22,23). We have also shown that this variant is a sequence context-dependent mutator, in that it induces mutations at a frequency 16 times higher than wild-type (WT) pol ␤ within a nucleotide sequence context found in the adenomatous polyposis coli gene that is frequently mutated in colon cancer (22,24).…”