Role of the tumor suppressor gene p53 in DNA polymerase beta-dependent base excision repair pathway.

摘要

Base excision repair (BER) pathway is a tightly coordinated sequence of events that results in the repair of a single damaged base and abasic sites. Emerging data point to p53 playing a potential role in regulating BER. This dissertation research aimed at investigating the role of p53 on beta-pol-dependent BER pathway. We hypothesized that p53 regulates DNA polymerase beta-dependent BER pathway via alteration in the expression of the rate limiting enzyme beta-pol. The level of BER capacity and expression of beta-pol and Ape1 were determined in HCT116 cells and in tissues derived from p53-deficient mice, at steady state and in response to DNA damage. In addition, we measured the effect of p53 gene dosage on accumulation of DNA damage. In the HCT116 colon cancer cells and in all tissues tested, we observed a significant decline in beta-pol mRNA and protein levels when p53 was deficient (P0.05). This decrease in beta-pol expression correlated with a deregulation in BER capacity. Additionally, p53 loss resulted in accumulation of endogenous abasic sites and this could contribute to the enhanced carcinogenesis observed in p53 null mice. The level of Ape1 protein at both steady state and in response to oxidative stress was unaffected by p53 status; however subcellular localization of Ape1 was dependent on p53. Furthermore, in response to DNA-damaging agents, the inducibility of BER and beta-pol was lost when p53 was deficient. More clearly, we have demonstrated that p53 deficiency results in increased DNA damage as a result of lack of inducibility of beta-pol and BER activity in response to cellular stress. We also determined the relationship between DNA polymerase beta expression and DNA replication in primary cells obtained from p53-deficient mice. beta-pol transcription in p53-deficient fibroblasts was independent of cellular growth; and serum deprivation served as stress signal for upregulation of beta-pol transcripts in wild-type cells. Thus, we provide evidence that p53 plays a role in regulating DNA polymerase beta expression at steady state and in response to oxidative stress both in vitro and in vivo; however, there exist tissue-specific differences in its regulation of the DNA polymerase beta-dependent BER pathway at steady state.