Assistant Professor, Dominican College of Blauvelt, NY
Regulation of DNA repair can be achieved through ubiquitin-mediated degradation of transiently induced proteins. In the budding yeast Saccharomyces cerevisiae, Rad4 is involved in damage recognition during nucleotide excision repair (NER) and, in conjunction with Rad23, recruits other proteins to the site of damage. Recent studies in budding yeast also suggest that the DNA damage surveillance pathways and mechanisms are tightly coordinated with mitotic and post-mitotic cell cycle events. The orchestration of these events is to ensure that when cells incur DNA damage, checkpoint signals are activated to induce cell cycle arrest thus allowing time for repair of the damaged DNA. A popular hypothesis is that these events are most appropriately sensed and mediated by proteins playing a dual role in these complex molecular processes.
This project evolved from a study that investigated the interaction of replication proteins Dbf4 and Cdc7, particularly in the context of interaction with the APC/C activator Cdc20. In a genome wide analysis to identify growth defects challenged with UV irradiation and other treatment modalities, we crossed 4700 non-essential yeast genes with cdc20-1, a thermolabile mutant protein that modulates the activity of the anaphase promoting complex (APC/C), a multisubunit E3 ubiquitin ligase complex. We serendipitously discovered a UV exposure-dependent interaction between Rad4 and Cdc20, as evident by the observation that the moderately UV sensitive Δrad4 strain became highly sensitive when cdc20-1 was present. This observation supports the hypothesis that the APC/CCdc20 plays a dual role coordinating APC/C activation and Rad4/Rad23-mediated NER.
We aim to determine the mechanistic relationship between Rad4 and Cdc20 with the goal of determining whether a direct or indirect interaction exists. The first step in identifying these other players is through an RNA-Seq analysis of single and double mutants when exposed to UV radiation. The significance of this study reaches beyond clarification of the role of proteolysis in DNA repair, but has practical implications in the treatments of certain cancers in which dysfunction of Cdc20 is suspected to play a role.
Materials are under development.
Materials are under development.
