For the elucidation of functionally important DNA polymerase beta (polbeta) variant, we propose experiments to test a hypothesis of whether a defective repair gene, such as polbeta, has a contributory role in the increased disease risks such as toxicity and carcinogenesis. A deletion of 87 bp in the cDNA of polbeta, a base-excision repair (BER) gene, in primary colorectal, breast, and lung tumors was reported from this laboratory. We demonstrated expression of both wild-type (WT) and truncated polbeta (polbetadelta) proteins in tumors. We established a stable murine fibroblast cell line, 16.3deltaP that expresses endogenous mouse WT and transfected human polbetadelta proteins. The BER and DNA binding activities in 16.3deltaP cells were markedly reduced compared to 16.3 parent cells, expressing the WT polbeta. The 16.3deltaP cells are hypersensitive to MNNG and MNU. Data showed that the 16.3deltaP nuclear extract inhibits BER and DNA binding activities of nuclear extract made from 16.3 cells. These results suggest that the polbeta variant may interfere with the functions of WT enzyme and act as a dominant negative mutant. These observations led us to examine the mechanisms underlying the inhibitory role of the polbetadelta in the functions of WT enzyme. Therefore, in the goal of Specific Aim 1, we propose to investigate potential hypersensitivity of 16.3deltaP cells when DNA is damaged by MNU evaluated by survival, susceptibility to transformation, ability to form colonies in semisolid medium and tumorigenic potential. Next we will investigate, in Specific Aim 2, the in vitro effect of the polbetadelta protein on the sensitivities of the transgenic F1 mice exposed to MNU. The proposed experiments will establish F1 mouse lines expressing the polbetadelta under the control of a mammary epithelial specific whey acidic protein promoter. A potential increase in occurrence of tumors in Fl mice treated with MNU and differences in the biochemical functions of polbeta between tumors and matched control tissues will be determined.
In Specific Aim 3, we will elucidate a possible mechanism of dominant negative activity of the polbetadelta by evaluating interactions of the polbetadelta protein with XRCC1, poly (ADP- ribose) polymerase and AP endonuclease, three repair proteins. These studies have the potential to gain insight into mechanisms by which truncated polbeta interferes with the functions of WT polbeta and would have broader implications into increased susceptibility of individuals to develop related diseases, toxicity, teratogenesis, and carcinogenesis, following exposure to environmental agents.
