RecA: Regulation and Mechanism of a Molecular Search Engine

Abstract

Homologous recombination maintains genomic integrity by repairing broken chromosomes. The broken chromosome is partially resected to produce single-stranded DNA that is used to search for homologous dsDNA. This homology-driven ‘search and rescue’ is catalyzed by a class of DNA strand exchange proteins that are defined in relation to Escherichia coli RecA, which forms a filament on single-stranded DNA. Here, we review the regulation of RecA filament assembly and the mechanism by which RecA quickly and efficiently searches for and identifies a unique homologous sequence amongst a vast excess of heterologous DNA. Given that RecA is the prototypic DNA strand exchange protein, its behavior affords insight into the actions of eukaryotic RAD51 orthologs and their regulators, BRCA2 and other tumor suppressors.

Protein splicing of a recombinase intein induced by ssDNA and DNA damage

“…Following splicing, T+1 is free to assist in MG 2+ -ATP binding, turning on ATPase activity. as has been shown for the better-studied bacterial RecA (Cox 2007;Bell and Kowalczykowski 2016) and eukaryotic Rad51 (San Filippo et al 2008;Krejci et al 2012), our work clearly demonstrates that ssDNA alone has a tremendous influence on the rate and accuracy of RadA splicing.…”

“…This work demonstrates a novel mechanism of recombinase control, adding an intriguing new example to the rich tapestry of recombinase regulation (Seitz et al 2001;Cox 2007;San Filippo et al 2008;Krejci et al 2012;Bell and Kowalczykowski 2016). DNA binding by RadA precursors is also of significance beyond our present study, given our discovery that host proteins may retain some activities prior to splicing.…”

“…To determine whether Pho RadA intein splicing can be influenced by intracellular ssDNA, we treated E. coli with nalidixic acid (Nal), a gyrase inhibitor that leads to ssDNA accumulation (Kohiyama et al 2013), or UV. Because induction of the SOS response generates the ssDNA acted on by recombinases during repair (Bell and Kowalczykowski 2016), we examined splicing in SOS + and their isogenic SOS − strain backgrounds (Supplemental Table 3). To this end, we used a deletion of the recA recombinase (recA − ) and a noncleavable variant of the lexA repressor (lexA3), both of which render the SOS response noninducible.…”

“…Such recombinases are ubiquitous in nature, being critical in DNA repair and homologous recombination (Seitz et al 2001;Lin et al 2006;Cox 2007;Bell and Kowalczykowski 2016). These proteins first bind ssDNA to form nucleoprotein filaments and then direct pairing with homologous dsDNA and initiate ssDNA strand invasion into the dsDNA template in an ATP-dependent manner (Seitz et al 2001).…”

A change of view: homologous recombination at single-molecule resolution

“…Bidirectional growth of RecA filaments was also observed in studies using fluorescently tagged RecA and a three -channel laminar flow system to control the assembly of RecA filaments on bead-tethered dsDNA molecules 23,86,87 (FIG. 3b).…”