The specific aim of this proposal is to isolate and characterize the proteins that constitute the damage-specific DNA incising activity involved in nucleotide excision repair in the yeast Saccharomyces cerevisiae. In order to isolate these proteins in the large amounts required for detailed biochemical studies we have cloned 4 of the 5 yeast RAD genes that are believed to code for the damage-specific DNA incising activity. We propose to isolate the remaining RAD gene by screening a yeast genomic library of plasmids containing yeast DNA inserts. We propose to subclone these RAD genes and to sequence all 5 genes of interest using the Maxan-Gilbert technique. We also propose to construct fusion plasmids in which the E. coli Beta galactosidase structural gene is under the control of RAD promoters. These fusion plasmids will be used to monitor induction of any RAD genes by measuring expression of Beta galactosidase following exposure of transformed (with single copy plasmids) cells to DNA damage. We also propose to tailor these fusion plasmids so as to replace the RAD promoters with a strong, regulable exogenous yeast promoter such as the GAL1 promoter, for over-expression of RAD proteins in yeast. The efficiency of the GAl1 promoter will be tested by measuring expression of Beta galactosidase in yeast. Over-expression of yeast RAD proteins will be achieved by deletion of the Beta galactosidase fragment. RAD proteins will be purified from transformed yeast cells and characterized biochemically and biophysically.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA012428-15
Application #
3163641
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1979-08-01
Project End
1989-04-30
Budget Start
1985-05-01
Budget End
1986-04-30
Support Year
15
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Stanford University
Department
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Reed, S H; Akiyama, M; Stillman, B et al. (1999) Yeast autonomously replicating sequence binding factor is involved in nucleotide excision repair. Genes Dev 13:3052-8
Reed, S H; You, Z; Friedberg, E C (1998) The yeast RAD7 and RAD16 genes are required for postincision events during nucleotide excision repair. In vitro and in vivo studies with rad7 and rad16 mutants and purification of a Rad7/Rad16-containing protein complex. J Biol Chem 273:29481-8
Rodriguez, K; Talamantez, J; Huang, W et al. (1998) Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein. J Biol Chem 273:34180-9
Wei, S; Friedberg, E C (1998) A fragment of the yeast DNA repair protein Rad4 confers toxicity to E. coli and is required for its interaction with Rad7 protein. Mutat Res 400:127-33
You, Z; Feaver, W J; Friedberg, E C (1998) Yeast RNA polymerase II transcription in vitro is inhibited in the presence of nucleotide excision repair: complementation of inhibition by Holo-TFIIH and requirement for RAD26. Mol Cell Biol 18:2668-76
Roush, A A; Suarez, M; Friedberg, E C et al. (1998) Deletion of the Saccharomyces cerevisiae gene RAD30 encoding an Escherichia coli DinB homolog confers UV radiation sensitivity and altered mutability. Mol Gen Genet 257:686-92
Ramos, W; Tappe, N; Talamantez, J et al. (1997) Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae. Nucleic Acids Res 25:1485-92
Wang, Z; Wu, X; Friedberg, E C (1997) Molecular mechanism of base excision repair of uracil-containing DNA in yeast cell-free extracts. J Biol Chem 272:24064-71
Feaver, W J; Henry, N L; Wang, Z et al. (1997) Genes for Tfb2, Tfb3, and Tfb4 subunits of yeast transcription/repair factor IIH. Homology to human cyclin-dependent kinase activating kinase and IIH subunits. J Biol Chem 272:19319-27
Wang, Z; Wei, S; Reed, S H et al. (1997) The RAD7, RAD16, and RAD23 genes of Saccharomyces cerevisiae: requirement for transcription-independent nucleotide excision repair in vitro and interactions between the gene products. Mol Cell Biol 17:635-43

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