Bacillus subtilis RadA/Sms-mediated nascent lagging-strand unwinding at stalled or reversed forks is a two-step process: RadA/Sms assists RecA nucleation, and RecA loads RadA/Sms

Abstract Replication fork rescue requires Bacillus subtilis RecA, its negative (SsbA) and positive (RecO) mediators, and fork-processing (RadA/Sms). To understand how they work to promote fork remodeling, reconstituted branched replication intermediates were used. We show that RadA/Sms (or its variant RadA/Sms C13A) binds to the 5′-tail of a reversed fork with longer nascent lagging-strand and unwinds it in the 5′→3′ direction, but RecA and its mediators limit unwinding. RadA/Sms cannot unwind a reversed fork with a longer nascent leading-strand, or a gapped stalled fork, but RecA interacts with and activates unwinding. Here, the molecular mechanism by which RadA/Sms, in concert with RecA, in a two-step reaction, unwinds the nascent lagging-strand of reversed or stalled forks is unveiled. First, RadA/Sms, as a mediator , contributes to SsbA displacement from the forks, and nucleates RecA onto single-stranded DNA. Then, RecA, as a loader , interacts with and recruits RadA/Sms onto the nascent lagging strand of these DNA substrates to unwind them. Within this process, RecA limits RadA/Sms self-assembly to control fork processing, and RadA/Sms prevents RecA from provoking unnecessary recombination.