Identification of Novel Core and Accessory Factors Involved in Nucleotide Excision Repair in Yeast

ABSTRACT Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism which deals with a wide variety of bulky, helix-distorting lesions, such as UV-induced cyclobutane pyrimidine dimers (CPDs). NER is traditionally grouped into two pathways: global genomic repair (GGR), which is operative throughout the genome, and transcription coupled repair (TCR), which is dedicated to rapid repair of the transcribed strand of actively transcribed genes. Though most of the core NER proteins are known, the exact biochemical mechanism of eukaryotic NER remains elusive. This dissertation focused on identifying novel core and accessory factors which function in NER. In the budding yeast Saccharomyces cerevisiae, GGR has previously been shown to be dependent on Rad7 and Rad16. We revealed Elc1, the yeast homolog of human elongin C, as a novel GGR-specific factor. Elc1 is required for GGR, but has no role in TCR. The precise role of Elc1 in GGR remains unknown. Dot1 is a histone methyltransferase whose sole substrate is histone H3 lysine 79 (H3K79). We identified Dot1 as another GGR-specific factor, as deletion of Dot1 or mutation of H3K79 abolishes GGR, but has no effect on TCR. H3K79 can accept up to 3 methyl groups, but Dot1 can only add one by itself. The PAF transcription elongation complex, through facilitating histone modifications, is partially required for dimethylation and fully required for trimethylation of H3K79 by Dot1. We demonstrated that through facilitating these histone modifications, PAF is partially required for GGR. TCR is believed to be triggered by a stalled elongating RNA polymerase II (Pol II) complex. Rad26, the homolog of the human CSB gene, and Rpb9, a nonessential subunit of Pol II, play important roles in TCR. We identified a dual role for PAF in TCR. In the presence of Rad26, PAF plays a positive role, facilitating TCR. In the absence of Rad26, PAF functions as a suppressor of TCR. PAF appears to be a part of a “megasuppressor” complex which includes Rpb4 and the Spt4/Spt5 complex, which also suppress Rad26-independent TCR. The interactions among Pol II, Rad26 and the various TCR suppressors remain to be elucidated