The long term goals are to elucidate the genetic and molecular mechanism of excision repair of DNA damaged by ultraviolet (UV) light irradiation in the yeast, Saccharomyces cerevisiae. The overall objective of this proposal is to examine the structure, regulation, and function of the RAD1, RAD2, RAD3, RAD4, and RAD10 genes and their encoded proteins involved in the incision step of excision repair, to reconstitute the incision activity in vitro, and to define the mechanism of its action. In addition, the viability role of the RAD3 gene and protein will be determined. The RAD4 gene is the only gene among these five genes that remains to be cloned; this gene will be isolated from the yeast genome by gap repair and its nucleotide sequence determined. The regulation of the RAD1, RAD2, RAD3, RAD4, and RAD10 genes and proteins will be examined following treatment of yeast cells with DNA damaging agents and during different cell cycle stages. The cellular location of the RAD1, RAD2, RAD3, RAD4, and RAD10 proteins will be determined by indirect immunofluorescence. The DNA dependent ATPASE and helicase activities of the RAD3 protein will be further characterized, and the role of RAD3 protein in recognition of the damaged site in DNA examined. To correlate the in vivo biological functions with the biochemical activities, various RAD3 mutant proteins will be overproduced and purified from yeast and biochemically characterized. The RAD1, RAD2, RAD4, and RAD10 proteins will be overproduced and purified from yeast cells and examined for DNA binding, ATPase, DNA unwindling, endonuclease and exonuclease activities. Combinations of RAD proteins will be examined for various biochemical activities and for incision of UV damaged DNA, and the mechanism of incision determined. To understand the essential role of the RAD3 gene in cell viability, heat sensitive (ts) mutations will be isolated and examined for effects on DNA replication, cell cycle, recombination, and other cellular processes. Genes coding for proteins that interact with the RAD3 protein in its viability function will be identified by isolating extragenic allele-specific cold sensitive (cs) suppressors of RAD3 ts mutations, and the biological roles of these cs suppressor genes determined. The wild type allele of cs suppressor genes will be cloned by complementation of the cs mutation and the structure, regulation, and function of these suppressor genes studied. Cells from xeroderma pigmentosum patients are defective in excision repair of UV induced pyrimidine dimers from DNA and these patients are prone to develop skin cancers, indicating a relationship between defective DNA repair and carcinogenesis. The proposed studies should provide a model system for understanding the mechanism of excision repair in eukaryotes, including in humans.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA035035-06
Application #
3172784
Study Section
Radiation Study Section (RAD)
Project Start
1983-07-01
Project End
1993-04-30
Budget Start
1988-07-01
Budget End
1989-04-30
Support Year
6
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Rochester
Department
Type
Schools of Arts and Sciences
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
Ribar, Balazs; Prakash, Louise; Prakash, Satya (2007) ELA1 and CUL3 are required along with ELC1 for RNA polymerase II polyubiquitylation and degradation in DNA-damaged yeast cells. Mol Cell Biol 27:3211-6
Ribar, Balazs; Prakash, Louise; Prakash, Satya (2006) Requirement of ELC1 for RNA polymerase II polyubiquitylation and degradation in response to DNA damage in Saccharomyces cerevisiae. Mol Cell Biol 26:3999-4005
Yu, Sung-Lim; Lee, Sung-Keun; Johnson, Robert E et al. (2003) The stalling of transcription at abasic sites is highly mutagenic. Mol Cell Biol 23:382-8
Lee, Sung-Keun; Yu, Sung-Lim; Prakash, Louise et al. (2002) Requirement of yeast RAD2, a homolog of human XPG gene, for efficient RNA polymerase II transcription. implications for Cockayne syndrome. Cell 109:823-34
Lee, Sung-Keun; Yu, Sung-Lim; Prakash, Louise et al. (2002) Yeast RAD26, a homolog of the human CSB gene, functions independently of nucleotide excision repair and base excision repair in promoting transcription through damaged bases. Mol Cell Biol 22:4383-9
Lee, S K; Yu, S L; Prakash, L et al. (2001) Requirement for yeast RAD26, a homolog of the human CSB gene, in elongation by RNA polymerase II. Mol Cell Biol 21:8651-6
Prakash, S; Prakash, L (2000) Nucleotide excision repair in yeast. Mutat Res 451:13-24
Habraken, Y; Sung, P; Prakash, L et al. (1998) ATP-dependent assembly of a ternary complex consisting of a DNA mismatch and the yeast MSH2-MSH6 and MLH1-PMS1 protein complexes. J Biol Chem 273:9837-41
Johnson, R E; Kovvali, G K; Prakash, L et al. (1998) Role of yeast Rth1 nuclease and its homologs in mutation avoidance, DNA repair, and DNA replication. Curr Genet 34:21-9
Habraken, Y; Sung, P; Prakash, L et al. (1997) Enhancement of MSH2-MSH3-mediated mismatch recognition by the yeast MLH1-PMS1 complex. Curr Biol 7:790-3

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