Characterization of the Replication Protein A (RPA)-70 Gene Family in Arabidopsis

University Of New Hampshire, Durham NH

Investigators

Abstract

Intellectual Merit: Replication Protein A (RPA), a eukaryotic single-stranded DNA-binding protein composed of three subunits (RPA70, 32 and 14), is required for almost every aspect of DNA maintenance, including DNA replication, repair, recombination, and cell-cycle checkpoint activation. Although much is known biochemically about RPA regulation in response to DNA damage, only limited genetic information is available about how RPA influences the complex molecular pathways of the DNA-damage response, since null mutations in RPA are generally not tolerated in animals and yeasts. To better understand the role of RPA in the DNA-damage response to double-strand breaks (DNA damage), the PI will genetically analyze mutants of a unique gene family of the large (RPA70) subunit of RPA in the model plant Arabidopsis thaliana. The PI hypothesizes that this gene family of 5 members represents an evolutionary division of RPA activity resulting from differences in how RPA is regulated in plants. "Knockout" T-DNA insertion mutations (null mutations) in each member of this gene family are tolerated, as are null mutations in two key regulators of the DNA-damage response, the protein kinases ATR and ATM. Therefore, the experiments outlined in this project provide a unique opportunity for a more straightforward genetic approach to understand RPA function in eukaryotic cells. The specific aims build upon preliminary data suggesting that individual RPA70 genes encode factors that participate in the DNA-damage response to double-strand breaks, perhaps by activating or regulating ATR and ATM. These specific aims include genetic characterization of RPA70 mutants to establish the roles of the individual proteins in regulating ATR and ATM, an analysis of RPA-dependent regulation of cell-cycle checkpoints and histone H2AX phosphorylation, and profiling of RPA70 expression in response to DNA damage. Broader Impacts: Both graduate and undergraduate students will play an active role in this research project. At the University of New Hampshire there are several research opportunities for undergraduates, including the INCO590 course that promotes underclassmen to participate in laboratory research. Over the course of the project, the PI will recruit multiple INCO590 participants to conduct aspects of the experiments. In addition, the PI will work with a UNH program called CONNECT, to provide a knowledge base of scientific opportunities on campus and to recruit minority underclassmen interested in science to participate in INCO590. Their research experience and training will help ensure a future generation of culturally diverse biological scientists for both public and private sectors.