Our long term objectives are to define the mechanisms of nucleotide excision repair (NER) in the yeast Saccharomyces cerevisiae.
The specific aims of this proposal are: (1) to identify the components of E3 ubiquitin ligase and to identify the E2 ubiquitin conjugating enzyme required for RNA polymerase II (Pol II) degradation in response to DMAdamage. The components of Eld -associated ubiquitin ligase will be identified and the physical and functional interactions of the E3 components with one another and with the E2 enzyme will be analyzed by genetic and biochemical means. The ubiquitin conjugation reaction on Pol II will be reconstitutedfrom purified components;(2) to delineate the role of Rad7-Rad16-Elc1 protein complex in translocation of polyubiquitinated Pol II to proteosome for degradation. The physical and functional interactions of Rad7, Rad16, and Eld with Pol II and with the proteosomal subunits will be analyzed by genetic and biochemical means;3) to identify the E2 and E3 enzymes required for Pol II monoubiquitination in response to DMA damage. Genetic studies will be done with mutations in these enzymes to reveal the physiological significance of this monoubiquitination event;(4) to determine the role of Psh1 in Rad6-Rad18 dependent histone ubiquitination and in chromatin remodeling during NER. The role of Rad6-Rad18-Psh1 complex in chromatin remodeling will be studied in biochemical experiments using a mononucleosomal DMA substrate containing a site specific DNA lesion and genetic studies will examine the role of these proteins in histone ubiquitination in responseto DNA damage in yeast cells;(5) to determine the role of Mms19 protein complex in NER and Pol II transcription. The role of this 4 subunit complex in NER and Pol II transcription will be studied by genetic and biochemical means;(6) to reconstitute NER on nucleosomal DNA. The NER system will be reconstituted on a mononucleosomal DNA substrate containing a site specific UV lesion and the role all the newly identified protein factors in NER will be determined. Defects in NER in humans cause the cancer-prone syndrome xeroderma pigmentosum and cells from patients with Cockayne syndrome, who suffer from severe growth and neurological problems, are defective in the repair of the transcribed DNA strand. We expect our studies in yeast to yield a comprehensive understanding of the underlying genetic bases of these human syndromes.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Okano, Paul
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University of Texas Medical Br Galveston
Schools of Medicine
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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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