Speaker
Description
DNA lesions ahead of replication forks can stall DNA replication. To maintain DNA fork stability and activate downstream DNA repair pathways, fork-remodeling enzymes process stalled forks into reversed forks. In humans, SMARCAL1 is thought to function at the early stage of fork reversal. Upon fork stalling, replication protein A (RPA), a high-affinity single-stranded DNA (ssDNA)-binding protein, binds to the exposed ssDNA as an initial response. Previous studies have shown that RPA specifically interacts with SMARCAL1, enhancing its activity on leading gap forks while inhibiting it on lagging gap forks.1,2 However, the detailed mechanism of this regulation remains unclear. To investigate this regulatory mechanism, we developed single-molecule fluorescence methods to direct monitor fork reversal, unwinding, and annealing using fluorescently labeled SMARCAL1 and RPA. We first reveal that, on leading-gap forks, RPA affects only the initiation stage of fork reversal, with no effect on subsequent four-way junction migration. Fluorescent SMARCAL1 exhibits highly dynamic association-dissociation on fork DNA. Kinetic analysis implies that SMARCAL1 binding involves more than one state. We classify SMARCAL1 binding into productive and non-productive states. In unwinding assays, we found that SMARCAL1 exhibits short-distance nascent strand unwinding activity, which is also regulated by RPA in a manner consistent with fork reversal. Annealing assays reveal that RPA does not significantly inhibit SMARCAL1-mediated DNA annealing on either leading- or lagging-gap forks. This study provides new insight that RPA regulates SMARCAL1 by modulating the population of productive and non-productive SMARCAL1 binding states, which may correlate with nascent strand unwinding activity.
