Rad54 Functions as a Heteroduplex DNA Pump Modulated by Its DNA Substrates and Rad51 during D Loop Formation

Abstract

Summary The displacement loop (D-loop) is the product of homology search and DNA strand invasion, constituting a central intermediate in homologous recombination (HR). In eukaryotes, Rad51 recombinase is assisted in D-loop formation by the Rad54 motor protein. Curiously, Rad54 also disrupts D-loops. How these opposing activities are coordinated toward productive recombination is unknown. Moreover, a seemingly disparate function Rad54 is removal of Rad51 from heteroduplex DNA (hDNA) to allow HR-associated DNA synthesis. Here, we uncover novel features of D-loop formation/dissociation dynamics, employing Rad51 filaments formed on ssDNAs that mimic physiological length and structure of in vivo substrates. The Rad54 motor is activated by Rad51 bound to synapsed DNAs and guided by a newly discovered single-stranded DNA binding domain. We present a unified model where Rad54 acts as an hDNA pump, which drives D-loop formation while simultaneously removing Rad51 from hDNA, consolidating both ATP-dependent activities of Rad54 into a single mechanistic step.

Efficient Detection of Unpaired DNA Requires a Member of the Rad54-Like Family of Homologous Recombination Proteins

“…During this process, a Rad51-coated single-stranded (ss) molecule of DNA invades a dsDNA template and searches for homology (Forget and Kowalczykowski 2012;Renkawitz et al 2013). A recently proposed model suggests that Rad54 aids this homology search in at least two ways (Wright and Heyer 2014): first, by driving together heteroduplex DNA, which consists of the invading strand and its complement within the original dsDNA template; and second, by dissociating the invading strand if it does not encounter a fully homologous complement. The close relationship between SAD-6 and Rad54 suggests that SAD-6 could promote homology identification in a similar manner.…”

Annealing of Complementary DNA Sequences During Double-Strand Break Repair inDrosophilaIs Mediated by the Ortholog of SMARCAL1

“…Recent work on the RPA-SMARCAL1 interface has shown that SMARCAL1 binds to the C-terminal region of RPA32 with 1:1 stoichiometry (Bhat et al 2014;Xie et al 2014) and it is likely that many molecules of Marcal1 bind the RPA-coated nascent DNA, allowing for multiple complementarity searches to occur simultaneously. Studies have revealed a similar mechanism for homology searching by Rad51-coated ssDNA (Wright and Heyer 2014;Qi and Greene 2016). Mutation of the Walker-B motif to allow ATP binding (thus preserving DNA-binding kinetics) but prevent ATP hydrolysis (translocation), as well as in vitro studies of Marcal1 interactions with long RPA-bound filaments, may help to clarify the mechanistic basis of the Marcal1 K275M phenotype.…”

DNA Strand Exchange and RecA Homologs in Meiosis

“…Biochemical studies have showed that Rad54 is capable of stripping Rad51 from the heteroduplex formed at sites of strand invasion (Kiianitsa et al 2006;Wright and Heyer 2014). Given their partial redundancy and similar phenotypes in vivo, it seems likely that Tid1/Rdh54 and Rad54 function similarly.…”

“…In the case of recombinogenic filament disassembly, homology between the donor and acceptor chromatids would allow the translocase to act as a "heteroduplex pump," which mechanistically couples the extension of heteroduplex DNA to stabilize the nascent joint molecule with removal of RecA homolog protomers from the heteroduplex DNA product (Fig. 4D,E) (Li and Heyer 2008;Wright and Heyer 2014). Finally, the DNA translocases may also contribute to the efficiency of repair by virtue of their nucleosome remodeling activity, which could also act to stabilize D-loops (Alexeev et al 2003;Zhang et al 2007;Hicks et al 2011).…”