Abstract
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 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 although 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 RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity.
Highlights
Replication protein A (RPA) inhibits SMARCAL1 translocation on some substrates but activates it on others
Normal replication forks containing RPA bound to the lagging-strand template are poor substrates for SMARCAL1catalyzed fork regression, whereas stalled forks with RPA bound to the leading-strand template are good substrates (Fig. 1B)
RPA bound to the chicken foot structures that are created by regression of stalled forks are poor substrates for SMARCAL1-catalyzed fork restoration, whereas chicken foot structures with RPA bound to the nascent leading strand are good substrates
Summary
Replication protein A (RPA) inhibits SMARCAL1 translocation on some substrates but activates it on others. SMARCAL1 interacts with replication protein A (RPA), the major ssDNA-binding protein (SSB) in human cells, and this interaction is required for SMARCAL1 localization (4 – 8). RPA confers substrate specificity to SMARCAL1, directing it to regress stalled forks caused by leading-strand lesions and to restore normal forks with laggingstrand ssDNA [15], consistent with a function for SMARCAL1 in promoting genome stability by catalyzing fork remodeling. RPA regulates the specificity of many other reactions during replication and repair while being rapidly placed on and taken off DNA [17, 23, 24] The mechanisms underlying this fundamental aspect of DNA biology remain largely unknown. We hypothesized that how RPA binds to the ssDNA in relation to the fork junction is important to create an optimal DNA-protein substrate for SMARCAL1. Our data support the second model and provide insights into how SSBs like RPA can generate substrate specificity for DNA translocases
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
