Abstract

The long term objectives are to define, in Saccharomyces cerevisiae, the roles of various nucleotide excision repair (NER) protein factors in damage recognition and in the assembly of the NER machinery at the damage site. The proposed studies will determine the role of the newly identified gene RFI1 in the repair of non-transcribed DNA and they will examine the damage binding properties of the purified Rfi1 protein. Genetic and biochemical studies will be performed to determine the role of the C4 and C3HC4 sequence motifs present in the Rfi1 protein and in the Rad16 protein. The physical and functional interaction of Rfi1 with the Rad7-Rad16 protein complex and with the other NER factors will be studied, and the role of the C3HC4 sequence present in Rfi1 and Rad16 on promoting association between these two proteins will be examined. The Abf1 protein is a component of the Rad7-Rad16 complex. The role of ABF1 in the repair of non-transcribed DNA will be examined and the damage binding properties of Abf1 protein will be studied. The Mms19 protein complex, which is comprised of four proteins of 100 (Mms19), 80, 55, and 43 kDa will be studied further. The identity of these Mmsl9 associated proteins will be determined by mass spectrometry, and genetic and biochemical studies will examine the role of these subunits and of the Mmsl9 complex in NER and in Pol II transcription. The order by which different NER factors assemble on damaged DNA will be analyzed by gel retardation and by DNase I footprinting. The role of the Rad7-Rad16 complex and of associated proteins in chromatin remodeling will be analyzed, and the effect of these proteins on the removal of UV lesions from nucleosomal DNA examined in the reconstituted system. Xeroderma pigmentosum (XP) patients are defective in NER of UV damaged DNA, and as a consequence, they suffer from a high incidence of skin cancers. Our studies in yeast should continue to provide important insights on the role of various protein factors in damage recognition and other steps of the NER process in eukaryotes, including in humans.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA041261-20
Application #
6835192
Study Section
Radiation Study Section (RAD)
Program Officer
Okano, Paul
Project Start
1985-12-01
Project End
2006-12-31
Budget Start
2005-01-01
Budget End
2005-12-31
Support Year
20
Fiscal Year
2005
Total Cost
$372,500
Indirect Cost

  Institution

Name
University of Texas Medical Br Galveston
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555

  Publications

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
Guzder, Sami N; Sommers, Christopher H; Prakash, Louise et al. (2006) Complex formation with damage recognition protein Rad14 is essential for Saccharomyces cerevisiae Rad1-Rad10 nuclease to perform its function in nucleotide excision repair in vivo. Mol Cell Biol 26:1135-41
Guzder, Sami N; Torres-Ramos, Carlos; Johnson, Robert E et al. (2004) Requirement of yeast Rad1-Rad10 nuclease for the removal of 3'-blocked termini from DNA strand breaks induced by reactive oxygen species. Genes Dev 18:2283-91
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
Johnson, Robert E; Yu, Sung-Lim; Prakash, Satya et al. (2003) Yeast DNA polymerase zeta (zeta) is essential for error-free replication past thymine glycol. Genes Dev 17:77-87
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
Unk, Ildiko; Haracska, Lajos; Gomes, Xavier V et al. (2002) Stimulation of 3'-->5' exonuclease and 3'-phosphodiesterase activities of yeast apn2 by proliferating cell nuclear antigen. Mol Cell Biol 22:6480-6
Unk, I; Haracska, L; Prakash, S et al. (2001) 3'-phosphodiesterase and 3'-->5' exonuclease activities of yeast Apn2 protein and requirement of these activities for repair of oxidative DNA damage. Mol Cell Biol 21:1656-61

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