High-affinity DNA Binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling [Enzymology]

December 31st, 2014 by Bhat, K. P., Betous, R., Cortez, D.

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with Replication Protein A (RPA), the major single-strand DNA (ssDNA) binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that while an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of the RPA DBD-A and DBD-B domains near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also find that the DBD-C and DBD-D RPA domains are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DNA binding domains at forks dictates SMARCAL1 substrate specificity.