Topological DNA-binding of structural maintenance of chromosomes-like RecN promotes DNA double-strand break repair in Escherichia coli

Abstract

Bacterial RecN, closely related to the structural maintenance of chromosomes (SMC) family of proteins, functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination. Here we show that the purified Escherichia coli RecN protein topologically loads onto both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) that has a preference for ssDNA. RecN topologically bound to dsDNA slides off the end of linear dsDNA, but this is prevented by RecA nucleoprotein filaments on ssDNA, thereby allowing RecN to translocate to DSBs. Furthermore, we found that, once RecN is recruited onto ssDNA, it can topologically capture a second dsDNA substrate in an ATP-dependent manner, suggesting a role in synapsis. Indeed, RecN stimulates RecA-mediated D-loop formation and subsequent strand exchange activities. Our findings provide mechanistic insights into the recruitment of RecN to DSBs and sister chromatid interactions by RecN, both of which function in RecA-mediated DSB repair.

SMC protein RecN drives RecA filament translocation and remodelling for in vivo homology search

“…Such negative feedback control by RecN could be direct, via influence of RecN on RecA turnover within the filament or indirect via modulation of the underlying DNA substrate [6, 7, 22]. This could drive RecA turnover at opposite ends of the filament [3, 5, 20, 21].…”

“…S5B is our proposed model of the same. Here, RecN action on the DNA could be a consequence of its reported bridging activity of ssDNA (generated around the break-site) with dsDNA 55 , thus driving directional movement of RecA, along with its underlying DNA substrate. 'Zipping' refers to RecN action ahead of the translocating RecA filament, while 'extrusion' refers to RecN action behind the filament 72,73 .…”

“…Studies have shown that RecA ATPase rates are stimulated 4-fold upon strand invasion, potentially allowing the RecA filament to release from heterologous pairing during microhomology sampling as well 43 . Separately, RecN has been found to (a) stimulate RecA strand invasion activity 49 and (b) bridge ssDNA and dsDNA 55 . Taken together, it is possible that the RecN-dependent RecA remodelling is a reflection of such microhomology sampling activity prior to homology pairing.…”

“…While their function in chromosome organization and segregation in bacteria has been well-characterized, their role in DNA repair pathways has not been clearly established. Previous studies have suggested a role for RecN in homologous recombination, either via its influence on kinetics of strand-invasion by the RecA filament or by facilitating global chromosomal cohesion between replicated sisters (20,22,25,26). Indeed the potential relevance of RecN in recombination-based repair is underscored by its extensive conservation with other core recombination-associated proteins such as RuvABC and RecA across bacterial genomes, as well as across domains of life (27).…”

Profiling a single-stranded DNA region within an rDNA segment that is a loading site for bacterial condensin

“…The physiological significance of the ssDNA-binding activity is currently not clear. However, several reports indicate that the ssDNA binding of the SMC proteins contributes to topological DNA binding on genomic DNA (Hirano and Hirano, 1998, 2006; Keyamura and Hishida, 2019; Murayama et al, 2018; Niki and Yano, 2016; Sutani and Yanagida, 1997; Sutani et al, 2015).…”