Living organisms employ multiple conserved pathways to repair different types of DNA lesions. One important pathway is nucleotide excision repair (NER), which is responsible for removal of bulky helix-distorting DNA lesions, such as UV induced photoproducts. A specialized NER pathway, called transcription coupled NER (TC-NER), refers to preferential repair in the transcribed strand of actively transcribed genes. In the yeast Saccharomyces cerevisiae, Rad26, the homologue of the human Cockaynes syndrome complementation group B protein, and Rpb9, a nonessential subunit of RNA polymerase II (Pol II), mediate two TC-NER mechanisms, respectively. Rpb4, another nonessential subunit of Pol II, plays a dual role in regulating TC-NER: suppressing Rpb9 mediated TC-NER and facilitating Rad26 mediated TC-NER. Recently, it was found that a third TC-NER mechanism that is independent of both Rad26 and Rpb9 but is normally suppressed by Spt4, a transcription elongation/suppression factor, also exists. At present, however, little is known about the molecular mechanism of TC-NER in eukaryotic cells. This project contains three objectives. Objective I is to examine interplay among Rad26, Rpb4, Spt4 and Pol II. The roles of Rad26 in regulating the cellular level of Spt4, and in modulating the association of Spt4 with Pol II will be analyzed. The role of Rpb4 in the loading of Rad26 to the Pol II complex following UV irradiation will be examined. The coordination among Rpb4 and Spt4 in their binding to core Pol II will be investigated. Objective II is to investigate the relationship between sumoylation of Rpb1, the largest subunit of Pol II, and TC-NER. Rpb1 is covalently modified by the small ubiquitin-like modifier (SUMO) in response to UV irradiation, and the modification is enhanced and persistent in TC-NER deficient cells. The role of Rpb1 sumoylation in TC-NER or in suppression of TC-NER will be examined by mutating the sumoylation sites on Rpb1. Objective III is to identify critical residues in the essential Pol II subunits that are implicated in TCR-NER. Ten out of twelve subunits (Rpb1 -12) of Pol II, i.e., those subunits other than Rpb4 and Rpb9, are essential for cell viability. The critical residues in these essential Pol II subunits that are involved in modulating TC-NER will be identified by isolating and characterizing Pol II mutants that are deficient in TC-NER or "super-proficient" in TC-NER.
Fulfillment of the proposed studies is expected to bring our understanding of the extremely complicated TC-NER mechanism in eukaryotic cells to a significantly higher level. Important findings generated from the proposed studies will not only be published in scientific journals, presented at local, national and international scientific meetings, but also be analyzed and interpreted in formats understandable to the non-scientist public. This project includes a significant educational component, with most of the funding used for research training of future professionals, including funding for lab members to present their data at scientific meetings. New methods developed and findings generated from the studies will be presented in the classroom. In addition, undergraduate research experiences will be emphasized as an integral part of the research.
DNA, the hereditary material in humans and almost all other organisms, is constantly being damaged by environmental factors and normal metabolic processes inside the cell. UV present in the sunlight is one of the major environmental DNA damaging sources. To contend with DNA damage, living organisms, including humans, employ multiple conserved DNA repair pathways. One important DNA repair pathway is nucleotide excision repair (NER), which is responsible for removal of bulky helix-distorting DNA lesions, such as UV induced photoproducts. A specialized NER pathway, called transcription coupled NER (TC-NER), refers to preferential repair in the transcribed strand of actively transcribed genes. Despite decades of intensive research, the molecular mechanism of TC-NER in eukaryotic cells is still largely elusive. Using the yeast as a model eukaryotic organism, we had demonstrated that Rad26, the homologue of the human Cockayne’s syndrome complementation group B protein, and Rpb9, a nonessential subunit of RNA polymerase II (Pol II), mediate two TC-NER mechanisms, respectively. Rpb4, another nonessential subunit of Pol II, plays a dual role in regulating TC-NER: suppressing Rpb9 mediated TC-NER and facilitating Rad26 mediated TC-NER. It had also been found that Spt4, a transcription elongation factor that forms complex with Spt5, suppresses TC-NER. This proposal was designed to answer specific questions concerning (1) how Pol II interplays with Rad26, Rpb4, Spt4 and Spt5 during TC-NER, (2) how sumoylation of Rpb1, the largest subunit of Pol II, is related to TC-NER, and (3) which residues in the essential Pol II subunits are involved in TC-NER or in the suppression of TC-NER. The project is relevant to an understanding of DNA repair in relation to cancer, premature and natural aging process, as well as the possible application of such understanding to more effective cancer therapy and extension of human life. We have fulfilled most, if not all, the goals set for the project and made breakthroughs in the field of DNA repair in eukaryotic chromatin. Studies supported by the award have resulted in 6 peer-reviewed publications, two book chapters, one invited review article and two PhD dissertations. The results have also been presented in 5 national or international scientific meetings. Multiple yeast strains, plasmids and experimental protocols have been created or developed, which has been and will continue to be shared with the scientific community. Importantly, this award has helped immensely for training future professionals. The findings we made have been used as teaching materials in the class room. Four graduate students, including a native American, 8 undergraduate students, including an African American, and 1 postdoctoral researcher were trained through this project.
