ROLES OF THE NUCLEIC ACID MOTOR PROTEIN ZGRF1 IN CHROMOSOME DAMAGE REPAIR

Exposure of cells to environmental agents, such as radiation, heavy metals, air pollutants andmutagenic chemicals, generates DNA double-strand breaks (DSB)s and other chromosomallesions, and can also cause replicative stress. Such environmentally induced chromosomallesions and stress are eliminated by a conserved mechanism - homologous recombination (HR).Defects in HR and its deregulation lead to genome destabilization, cancer, and other diseases.Several nucleic acid motor proteins, including helicases, have been implicated in mechanismsthat affect HR outcome and DNA damage repair, relieve cells from replication fork stress, andmediate the resolution of R-loops.ZGRF1, a nucleic acid motor, has been implicated in HR and DNA crosslink repair in siRNA-based genomic screens. We have found that engineered ZGRF1-/- cells are hypersensitivity tomitomycin C (MMC) treatment and accumulate chromosome aberrations upon drug treatment.Importantly, purified ZGRF1 shows DNA dependent ATPase, D-loop and R-loop dissociation, andreplication fork regression activities, suggesting that ZGRF1 functions directly to regulate HR andmediate replication fork repair and R-loop resolution. In this project, we will apply our considerableexpertise in molecular studies of nucleic acid motor proteins to define the mechanisms by whichZGRF1 accomplishes its biological functions. In Aim 1, we will perform a variety of genetic andcytological studies to examine ZGRF1 mutant cells for defects in DNA damage repair, replicationfork maintenance/repair, and also R-loop resolution. We will ascertain the biological relevance ofthe ZGRF1 nucleic acid motor activity with an ATPase defective mutant that we have generated.In Aim 2, we will define the biochemical attributes of ZGRF1, and carry out co-immunoprecipitation and biochemical pulldown to identify interactors of this motor protein. In Aim3, we will investigate the biochemical and genetic defects of ZGRF1 mutants altered in the zincfinger domain, impaired for protein-protein interactions, or found in cancer. The results from ourproject will shed light on the roles of ZGRF1 in nuclear processes that are germane for delineatinghow disease causative mutations and chromosome rearrangements arise in cells deficient innucleic acid motors. The results on R-loop resolution are expected to contribute toward thedevelopment of novel strategies to avoid the accumulation of R-loops upon exposure toenvironmental stress and mutagens.