DNA Polymerase b (pol b) produces a nuclear protein important in short gap filling during DNA repair. Early reports using tumor cDNA suggested that pol b is mutated in several human cancers. However, subsequent reports suggested these changes may actually be alternative splice variants of pol b. We tested the hypothesis that Pol b is frequently mutated in bladder and lung cancers and that splice variants and inherited polymorphisms in this gene may affect susceptibility to bladder and lung cancers. Using SSCP analysis we screened for pol b mutations in exons 1 through 14, the promoter region and exon a using genomic DNA from human bladder cancer cell lines, primary bladder tumors and primary lung tumors. We discovered a CCC to CGC transversion at codon 242 in one bladder cancer cell line that results in a proline to arginine amino acid substitution, which we have subsequently demonstrated to be a polymorphism. We have demonstrated this polymorphism in normal DNA from 7 of 180 bladder cancer cases and 6 of 162 controls. In collaboration with Dr. Sam Wilson, we have looked at the expression and activity of the codon 242 polymorphism in a heterozygous cell line, J82. Using an activity assay specific for pol b's role in BER, we found J82 to have reduced repair activity while expressing close to normal amounts of the protein. Through mixing experiments, we found J82 lysate to suppress, in a linear fashion, the normal BER activity of ScaBer, a cell line with wildtype pol b suggesting that the variant may have dominant negative activity. We are continuing investigations in collaboration with the Wilson lab by transfecting the codon 242 polymorphism into a normal mouse fibroblast cell line null for pol b to characterize activity. By screening normal DNA from bladder cancer cases and controls, additional polymorphisms have been found in the promoter region (7/90) and in exon 1 (1/87). These are presently being sequenced. Also ongoing is analysis of different splice variants of pol b by cloning them into a vector and sequencing them. We have identified 6 different splice variants so far in human bladder cancer cell lines, most of which have never been reported. We are investigating the different splicing found in human bladder tumor and normal bladder matched pairs.