A variant of DNA polymerase beta acts as a dominant negative mutant.

Abstract:

In eukaryotic cells, DNA polymerase beta (polbeta) carries out base-excision repair (BER) required for DNA maintenance, replication, recombination, and drug resistance. A specific deletion in one allele in the coding sequence of the polbeta gene occurs in colorectal and breast carcinomas. The 87-bp deleted region encodes amino acid residues 208-236 in the catalytic domain of the enzyme. Here, we report evidence for expression of the wild-type (WT) and the truncated polbeta proteins in colorectal tumors. To elucidate the potential functional consequences of polbeta truncation, stable HeLa cell lines were established from cloned WT and variant polbetaDelta208-236. Cells expressing the variant protein exhibited substantially decreased BER activity. To test our hypothesis that truncated polbeta may disrupt the function of the WT enzyme, we stably transfected mouse embryonic fibroblast 16.3 cells with polbetaDelta208-236 cDNA. Reverse transcription-PCR and Western blot analyses showed that the new cell line, 16.3DeltaP, expresses the WT and the truncated polbeta mRNA and proteins. BER and binding activities were undetectable in these cells. Furthermore, in vivo the 16.3DeltaP cells were more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine than the 16.3 cells. On adding increasing amounts of 16.3DeltaP protein extracts, the BER and DNA binding activities of extracts of the parent 16.3 cell line progressively declined. These results strongly suggest that truncated polbeta acts as a dominant negative mutant. The defective polbeta may facilitate accumulation of mutations, leading to the expression of a mutator phenotype in tumor cells.

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