Cancer is a leading cause of death and morbidity. It results from the accumulation of genetic mutations that ultimately lead to a growth advantage and expansion of rare cellular clones. Understanding the etiology of genetic mutation is thus of tremendous importance. Fundamentally, mutations represent the failure of a cell to correctly repair the DNA lesions caused by a variety of endogenous and exogenous damaging agents. This proposal is based on the model that inefficient repair of DNA double-strand breaks (DSBs) leads to persistence of lesions that ultimately become substrates for chromosomal rearrangement, a hallmark of malignancy. It focuses specifically on the enzymes that process DSB ends to make them ready for ligation, since failure of these enzymes might contribute substantially to persistence of DSB lesions in recombinogenic forms. All experiments use Saccharomyces cerevisiae as a model organism. State-of-the-art genomic tools are used to study many yeast genes in parallel and in combination, which is critical since multiplicity and redundancy of end processing pathways is anticipated. The first Specific Aim is to identify enzymes that resect the 5'-terminated strand in homologous recombinational repair (HRR). Preliminary evidence suggests that these cause an efficient and irreversible commitment to HRR in budding yeast. Novel competitive assays are based on the hypothesis that impaired 5' resection will increase the contribution of nonhomologous end-joining (NHEJ) and the likelihood of chromosomal rearrangement. High probability candidate helicases and nucleases will be examined in detail, in addition to panel and mutational screens for other involved genes. The second Specific Aim is to elucidate the mechanism of POL4 (yeast DNA polymerase b)-dependent processing in NHEJ. The hypothesis that this gene's previously described role represents an overlap with base excision repair is examined by chimeric analysis with the human enzyme. Protein interaction, panel and mutational screens will be used to identify Po14p-interacting nucleases. The third Specific Aim is to describe the processing of 5' hydroxyl and 3' phosphate terminal DSB lesions. A novel plasmid transformation assay will be used to explore the extent and mechanism of repair. This assay, in vitro biochemical assays, and cellular responses to chemical mutagens will evaluate the hypothesis that ORF YMR156c is the 3' phosphatase portion of yeast polynucleotide kinase.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA090911-03
Application #
6634030
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Pelroy, Richard
Project Start
2001-04-06
Project End
2005-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
3
Fiscal Year
2003
Total Cost
$213,038
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pathology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Daley, James M; Wilson, Thomas E (2005) Rejoining of DNA double-strand breaks as a function of overhang length. Mol Cell Biol 25:896-906
Daley, James M; Laan, Renee L Vander; Suresh, Aswathi et al. (2005) DNA joint dependence of pol X family polymerase action in nonhomologous end joining. J Biol Chem 280:29030-7
Karumbati, Anandi S; Wilson, Thomas E (2005) Abrogation of the Chk1-Pds1 checkpoint leads to tolerance of persistent single-strand breaks in Saccharomyces cerevisiae. Genetics 169:1833-44
Deshpande, Rajashree A; Wilson, Thomas E (2004) Identification of DNA 3'-phosphatase active site residues and their differential role in DNA binding, Mg2+ coordination, and catalysis. Biochemistry 43:8579-89
Karumbati, Anandi S; Deshpande, Rajashree A; Jilani, Arshad et al. (2003) The role of yeast DNA 3'-phosphatase Tpp1 and rad1/Rad10 endonuclease in processing spontaneous and induced base lesions. J Biol Chem 278:31434-43
Karathanasis, Elissa; Wilson, Thomas E (2002) Enhancement of Saccharomyces cerevisiae end-joining efficiency by cell growth stage but not by impairment of recombination. Genetics 161:1015-27
Vance, John R; Wilson, Thomas E (2002) Yeast Tdp1 and Rad1-Rad10 function as redundant pathways for repairing Top1 replicative damage. Proc Natl Acad Sci U S A 99:13669-74
Wilson, Thomas E (2002) A genomics-based screen for yeast mutants with an altered recombination/end-joining repair ratio. Genetics 162:677-88