Base excision repair is a major mechanism to correct DNA damage, such as modified bases and apurinic/apyrimidinic (AP) sites. These lesions are among the most abundant lesions generated through normal cellular metabolisms and are also induced by environmental agents including many anti-cancer drugs and ionizing radiation. In addition, many of these lesions are mutagenic if not repaired. Thus, understanding the mechanism of base excision repair is an urgent subject for reducing the cancer risk and also for improving cancer treatments. The long-term objective of this research project is to define the fundamental reaction of base excision repair in higher eukaryotes. The first specific aim is to elucidate the excision mechanism of a 5'- incised AP site by DNA polymerase beta (pol beta) from structural and functional perspectives. Three approaches will be made toward this goal: 1) functional analyses of a series of site-directed mutations introduced into residues in the N-terminal 8-kDa domain of pol beta which is responsible for the excision activity; 2) stereochemical analyses of the DNA substrate and the product of this excision reaction; 3) crystallographic analyses of the 8-kDa domain/DNA complexes. Since the 8-kDa domain contains a helix-hairpin-helix motif, a DNA-binding motif which has been found in numbers of proteins involved in DNA transaction, the information obtained from this study will also contribute to understanding the interaction of these proteins with DNA. The second specific aim in this project is to dissect a protein complex assembled on an AP site during the PCNA-dependent repair reaction which presumably serves as an alternative pathway of base excision repair in higher eukaryotes in addition to the pol beta-dependent pathway. Since previous studies suggest the importance of the structure of the DNA substrate (circular DNA versus linear DNA) in the PCNA-dependent reaction in vitro, the effect of end-capping of linear DNA on the assembly of the PCNA-dependent repair complex will be evaluated. Furthermore, the roles of replication factor C (RF-C) and PCNA in recognition of a 5'-incised AP site and interaction with DNA, DNA polymerase delta, flap endonuclease 1 (FEN1) and DNA ligase in the repair complex will be characterized in a reconstituted system. Some of the protein factors employed in the PCNA-dependent AP site repair are also involved in DNA replication and nucleotide excision repair. However, since the 5'-incised AP site is a unique DNA structure in this repair system, this study will add a new insight to the functions of these versatile proteins.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Chemical Pathology Study Section (CPA)
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Okano, Paul
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Fox Chase Cancer Center
United States
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