Base excision repair is the major mechanism for correcting modified bases, abasic sites and single-strand breaks. These DNA lesions are generated during normal metabolic processes through free radicals and reactive chemical agents. They are also generated by several chemotherapeutic drugs and ionizing radiation and contribute to the normal tissue toxicities associated with these treatments. A comprehensive understanding of this repair mechanism is important for improving cancer prevention and treatment programs. In the past, base excision repair was thought to proceed exclusively by a DNA polymerase beta (pol beta)-dependent pathway. My laboratory, was first to identify an alternative pathway which requires proliferating cell nuclear antigen (PCNA) as an essential factor. Analysis of in vivo DNA repair also indicated that damage generated by gamma-irradiation and hydrogen peroxide, but not by an alkylating agent, was efficiently repaired in pol beta-knockout cells, suggesting that the PCNA-dependent pathway is functional in vivo. PCNA plays an essential role in important cellular functions: DNA replication, cell cycle control and DNA repair, and physically interacts with replication factor C, DNA polymerases delta and epsilon, flap endonuclease 1, p2lWaf1/Cip1, D-type cyclins, and Gadd45. p2l is induced by DNA damage and consequently arrests cell-cycle progression in G1. The current dogma is that cell-cycle arrest will provide enough time for DNA repair before cells replicate their DNA in S phase. However, our in vitro studies indicate that the PCNA-dependent base excision repair is inhibited by p2l. The overall goal of our research is to determine the relative importance of base excision repair in cancer prevention and therapy. In this project, we will elucidate how the PCNA-dependent pathway of base excision repair is regulated through interactions of PCNA with p2l, D-type cyclins, and Gadd45. To address this question, [Aim 1] we will introduce PCNA, p21, cyclin D1, and a dominant negative mutant of pol beta on an inducible expression vector into cultured cells and analyze the effects of their overexpression on base excision repair and control of cell-cycle checkpoints; [Aim 2] we will investigate the physiological relevance of p2l induction to base excision repair in various cell lines which exhibit different induction levels of p2l; [Aim 3] we will introduce a series of mutant PCNA which are defective in interactions with specific factor(s) into cultured cells and analyze the effects of their overexpression on base excision repair and control of cell-cycle checkpoints.
| Hayashi, Harutoshi; Tominaga, Yohei; Hirano, Seiki et al. (2002) Replication-associated repair of adenine:8-oxoguanine mispairs by MYH. Curr Biol 12:335-9 |
