Examining Homologous Recombination through the Biochemical Characterization of Eukaryotic Recombinases and Accessory Proteins

Andrew A. Kelso, Clemson University

Abstract

Homologous recombination (HR) is a template-driven repair pathway that helps to maintain genome stability by mending DNA double-stranded breaks (DSBs) and to create genetic diversity in progeny during meiosis. The formation of a DSB is highly toxic to the cell because a single DSB can result in cell death. The majority of the HR repair machinery is conserved in higher eukaryotes and defects in the repair proteins can result in the inability to repair, or improper repair of DSBs, leading to genome instability and cancers. Also, errors in meiotic recombination such as improper segregation or non- allelic recombination are associated with genetic disorders. When a DSB occurs, the break site is nucleolytically processed to yield a 3' ssDNA overhang that is essential for the nucleation of the recombinase—the central enzyme of HR. The recombinase forms a right-handed, helical nucleoprotein filament (also know as the presynaptic filament) that is responsible for homology search within a homologous template. RAD51 is the recombinase that performs homology search and DNA joint molecule formation in somatic cells, and DMC1 is the meiosis-specific recombinase that facilitates the exchange of genetic information in meiosis. The HR pathway relies on the critical functions of the recombinases, but also requires many accessory proteins. Here, I present data demonstrating functional Rad51 and Dmc1 recombinases in the protozoan parasite Entamoeba histolytica—an organism in which HR is largely uncharacterized. Additionally, I provide new evidence for the functions of a few recombinase accessory proteins and their role in HR. We show that the BRCA2 interacting partner, BCCIPβ is able to enhance the recombinase activities of RAD51 through the induction of conformational changes within the RAD51 filament that promote the release of ADP, helping to keep RAD51 active. Also, using an ATP binding-deficient variant of DMC1, we demonstrate that the meiosis-specific protein complex Hop2-Mnd1 stimulates the activities of DMC1 by promoting nucleotide binding. And lastly, we demonstrate that the epigenetic modulator HELLS has a function in HR as an ATPase-independent stimulator of RAD51 and an ATPase-dependent DNA branch migrator. The findings presented here provide novel molecular functions for eukaryotic recombinases and accessory proteins involved in HR, and therefore, advance the understanding of the mechanisms that contribute to genome stability.