RADX protein interactions regulate replication fork stability

Abstract

Genome integrity requires complete and accurate DNA replication once per cell division cycle. Replication stress poses obstacles to this process that must be overcome to prevent replication fork collapse. An important regulator of replication fork stability is the RAD51 protein, which promotes replication fork reversal and protects nascent DNA strands from nuclease-mediated degradation. In this dissertation, I characterize the importance of RADX interaction with itself and RAD51 at replication forks. In Chapter III, I demonstrate that a direct protein-protein interaction between RADX and RAD51 is essential for replication fork stability and genome integrity. In chapter IV, I identify and characterize RADX oligomerization and assess its importance for RADX function at replication forks. Using biochemical and genetic approaches, we found that RADX acts as a homo-oligomer to control replication fork stability. RADX oligomerizes using at least two different interaction surfaces, including one mapped to a C-terminal region. We demonstrate that mutations in this region prevent oligomerization and prevent RADX function in cells, and that addition of a heterologous dimerization domain to the oligomerization mutants restored their ability to regulate replication. Taken together, our results demonstrate that like many ssDNA-binding proteins, oligomerization is essential for RADX-mediated regulation of genome stability.