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.
The DNA damage response is a complex array of molecular pathways to sense and respond to persisting DNA damage, thereby mitigating the threat of mutation. Downstream responses include transcriptional and post-transcriptional changes, induction of repair pathways, cell-cycle checkpoint regulation, and in some cases apoptosis (cell death). These response pathways are ultimately regulated by proteins that physically interact with DNA, to recognize lesions or replication blocks. A major player in this sensing of DNA damage is the single-stranded DNA binding protein Replication Protein A (RPA). RPA is composed of three subunits, RPA70, -32, and -14, forming a heterotrimer that physically binds single-stranded DNA. The model plant Arabidopsis thaliana encodes five conserved RPA70 subunits, three of which are induced by ionizing radiation (IR), and mutations in at least two of these subunits confers hypersensitivity to a range of DNA damaging agents. In comparison, yeasts and animals typically contain only a single gene for each subunit, and mutations in these genes are lethal. RPA is phosphorylated in response to DNA damage, and this phosphorylation is believed to play roles in the regulation of RPA activities, including a switch from its normal role in DNA replication to a modified role to activate the DNA damage response. In light of the additional subunits encoded in Arabidopsis, the PI hypothesizes that plants use additional forms of RPA regulation in place of, or in cooperation with, phosphorylation-dependent regulation found in animal cells. The overarching goal of this project has been to better understand how eukaryotic (animal and plant) cells employ signaling proteins that directly interact with DNA to regulate DNA damage responses. The specific aims of this project included characterization of individual RPA70 subunit regulation in response to DNA damage, and genetic interactions of RPA70 gene family members with two master regulators of the DNA damage response, ATR and ATM. We have found that individual RPA70 subunit proteins have unique roles in the DNA damage response. Through our expreiments, we have shown that defects in RPA70 genes confer different physical (phenotypical) traits in response to a variety of agents that damage DNA. In one case, we have identified a RPA subunit gene (RPA70C) that perhaps negatively regulates DNA replication in response to DNA damage. This is important since this may define a unique checkpoint, or regulatory mechanism, that prevents downstream mutation within plant tissues and the germline. Due to their sessile nature and metabolic requirements, plants are constantly under attack by DNA damaging agents, such as UV light and oxidative damage that can lead to double-strand breaks, and ultimately cell death. Utilizing genetic and biochemical approaches to understand RPA functions, these experiments will provide much needed insight into how plants respond and cope with DNA damage, potentially leading to improved crop species. In addition, Arabidopsis represents a unique genetic system in which null mutations in genes involved in the DNA damage response (such as RPA) are tolerated. As a result, these studies in Arabidopsis have the potential to reveal novel DNA damage-response mechanisms also present in animals (humans). The proposed work provided significant research training in advanced biochemical, genetic, and genome biology to six undergraduate student and two graduate student investigators. The award also employed a research technician and multiple undergraduate students for summer jobs to help offset tuition costs etc.
