The objective of this proposal is to determine the function of RAD52 in mammalian somatic cells. Extensive genetic evidence implicates the RAD52 gene in a recombinational DNA repair pathway in the yeast Saccharomyces cerevisiae. Mutations that inactivate this gene render yeast strains exquisitely sensitive to DNA damaging agents, and dramatically reduce the frequency of nearly all forms of cellular homologous recombination. Murine and human homologs of this gene have been identified, and shown to be expressed in somatic tissue. The principal investigator and others have shown that overexpression of either human or yeast alleles of this gene in mammalian system renders the host cells resistant to DNA damaging agents, while simultaneously enhancing the frequency of cellular homologous recombination. In addition, the PI have determined that overexpression of human antisense RAD52 mRNA renders human fibrosarcoma cells sensitive to a number of physical and chemical DNA damaging agents. The PI believes that these observations support the hypothesis that a RAD52 recombinational repair pathway functions in mammalian somatic cells. One of the major objectives of this proposal is to test this hypothesis by creating mammalian somatic cells that lack a functional RAD52 gene. Similar RAD52-knockout experiments will be performed in cell lines deficient in DNA-dependent protein kinase activity. The PI suggests that these latter experiments will permit him to evaluate the relative importance of the recombinational and end-joining repair pathways in mammalian cells, and allow him to test the hypothesis that these two apparently distinct repair pathways genetically interact. In addition, the PI intend to identify and clone mammalian Rad52-binding proteins. The objective of this strategy is to both gain insight into the exact function of the Rad52 protein, as well as to further dissect the RAD52-dependent recombinational repair process in mammalian cells. There has been a great deal of interest in the role of DNA repair in human cancer of late. Recent developments have shown that the link between deficient DNA repair and cancer is more extensive than had been previously believed. It has also become clear that tumor cells may utilize enhanced DNA repair activity to evade the cytotoxic effects of chemotherapeutic agents. A greater understanding of the function of RAD52-dependent DNA repair in mammalian somatic cells is therefore likely to have important implications for the fields of carcinogenesis and anti-cancer therapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29CA061906-01A4
Application #
2008371
Study Section
Biological Sciences 2 (BIOL)
Project Start
1997-02-01
Project End
2002-01-31
Budget Start
1997-02-01
Budget End
1998-01-31
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Pharmacology
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Bordone, Laura; Campbell, Colin (2002) DNA ligase III is degraded by calpain during cell death induced by DNA-damaging agents. J Biol Chem 277:26673-80
Lakshmipathy, U; Campbell, C (2001) Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity. Nucleic Acids Res 29:668-76
Lundberg, R; Mavinakere, M; Campbell, C (2001) Deficient DNA end joining activity in extracts from fanconi anemia fibroblasts. J Biol Chem 276:9543-9
Donahue, S L; Corner, B E; Bordone, L et al. (2001) Mitochondrial DNA ligase function in Saccharomyces cerevisiae. Nucleic Acids Res 29:1582-9
Lakshmipathy, U; Campbell, C (2000) Mitochondrial DNA ligase III function is independent of Xrcc1. Nucleic Acids Res 28:3880-6
Coffey, G; Campbell, C (2000) An alternate form of Ku80 is required for DNA end-binding activity in mammalian mitochondria. Nucleic Acids Res 28:3793-800
Marsh, T C; Cole, E S; Stuart, K R et al. (2000) RAD51 is required for propagation of the germinal nucleus in Tetrahymena thermophila. Genetics 154:1587-96
Coffey, G; Lakshmipathy, U; Campbell, C (1999) Mammalian mitochondrial extracts possess DNA end-binding activity. Nucleic Acids Res 27:3348-54
Lakshmipathy, U; Campbell, C (1999) The human DNA ligase III gene encodes nuclear and mitochondrial proteins. Mol Cell Biol 19:3869-76
Lakshmipathy, U; Campbell, C (1999) Double strand break rejoining by mammalian mitochondrial extracts. Nucleic Acids Res 27:1198-204

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