Mechanism of strand exchange from RecA-DNA synaptic and D-loop structures.

Abstract

The strand exchange reaction is central to homologous recombination. It is catalyzed by the RecA family of ATPases that form a helical filament with single-stranded DNA (ssDNA) and ATP. This filament binds to a donor double-stranded DNA (dsDNA) to form synaptic filaments that search for homology, and then catalyze the exchange of the complementary strand to form a new heteroduplex, or a D-loop if homology is limited1,2. Here we report the Cryo-EM analysis of synaptic mini filaments with both non-complementary and partially-complementary dsDNA, and structures of RecA–D-loop complexes containing a 10 or 12 base pair heteroduplex at 2.8 and 2.9 Å, respectively. The RecA C-terminal domain (CTD) binds to dsDNA and directs it to the L2 loop, which inserts into and opens the duplex. The opening propagates through RecA sequestering the homologous strand at a secondary DNA-binding site, freeing the complementary strand to sample pairing with the ssDNA. Duplex opening has a significant probability of stopping at each RecA step, with the as yet unopened dsDNA portion binding to another CTD. Homology suppresses this process through heteroduplex pairing cooperating with secondary site-ssDNA binding to extend dsDNA opening. This mechanism locally limits the length of ssDNA sampled for pairing if homology is not encountered, and it may provide for the formation of multiple synapses separated substantially on the donor dsDNA, increasing the probability of encountering homology.