Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution

Abstract

During recombinational repair of double-stranded DNA breaks, RAD51 recombinase assembles as a nucleoprotein filament around single-stranded DNA to form a catalytically proficient structure able to promote homology recognition and strand exchange. Mediators and accessory factors guide the action and control the dynamics of RAD51 filaments. Elucidation of these control mechanisms necessitates development of approaches to quantitatively probe transient aspects of RAD51 filament dynamics. Here, we combine fluorescence microscopy, optical tweezers, and microfluidics to visualize the assembly of RAD51 filaments on bare single-stranded DNA and quantify the process with single-monomer sensitivity. We show that filaments are seeded from RAD51 nuclei that are heterogeneous in size. This heterogeneity appears to arise from the energetic balance between RAD51 self-assembly in solution and the size-dependent interaction time of the nuclei with DNA. We show that nucleation intrinsically is substrate selective, strongly favoring filament formation on bare single-stranded DNA. Furthermore, we devised a singlemolecule fluorescence recovery after photobleaching assay to independently observe filament nucleation and growth, permitting direct measurement of their contributions to filament formation. Our findings yield a comprehensive, quantitative understanding of RAD51 filament formation on bare single-stranded DNA that will serve as a basis to elucidate how mediators help RAD51 filament assembly and accessory factors control filament dynamics.ouble-stranded DNA (dsDNA) breaks are severe forms of genetic lesions that may result in chromosome instability (1-3). Organisms have devised several pathways to mend dsDNA breaks. Among these, recombinational repair mediated by bacterial RecA-like ATP-dependent recombinases is the most accurate, because it is capable of restoring chromosome integrity without loss of genetic information (2, 4). During recombinational repair in humans, broken dsDNA ends are first resected to create single-stranded DNA (ssDNA) overhangs that are coated quickly by replication protein A (RPA). The ATP-dependent recombinase protein RAD51, the focus of this study, must next compete with RPA to assemble nucleoprotein filaments around these ssDNA overhangs. These filaments form the structures that can promote homology recognition in an intact homologous duplex and catalyze DNA strand exchange, resulting in joint molecule intermediates. After RAD51 disassembly from the heteroduplex DNA, the invading strand can prime DNA synthesis to recover lost genetic information. RAD51, however, does not act alone during recombinational repair. Mediators and accessory factors stringently control the dynamics of RAD51 filaments by acting at the level of formation, stabilization, or even disassembly of these filaments (2,3,5). One important level of control occurs at the assembly of nascent RAD51 filaments on RPA-coated ssDNA. On its own, RAD51 cannot load on the RPA-coated substrate but requires the action of a mediator to guide an...

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

“…The observed disassembly rates of (14 ± 2) 10 −4  s −1 (at 5 pN; N  = 8; dissociation rates are obtained from exponential fits to the fluorescence intensity traces, errors are fitting errors), (15 ± 1) 10 −4  s −1 (at 20 pN; N  = 16) and (12 ± 1) 10 −4  s −1 (at 50 pN; N  = 4) show that hRAD51 dissociation from ssDNA is not influenced by tension within the range of values tested. Interestingly, a similar effect was reported previously (Candelli et al , 2014) for the assembly process: both nucleation and growth of hRAD51 NPFs are highly tension‐dependent for dsDNA, but independent of tension for ssDNA. This finding can be attributed to the fact that dsDNA is more rigid and resistant to length change (King et al , 2013).…”

“…Our previous single‐molecule work on the interaction of hRAD51 with ssDNA focused mainly on the assembly of the NPF (Candelli et al , 2014) and on the disassembly of hRAD51 from double‐stranded DNA (dsDNA; van Mameren et al , 2009b). The latter study showed that hRAD51 disassembles through a pause–burst mechanism from NPF ends, dominated by ATP hydrolysis of the RAD51 monomers at filament ends.…”

Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics

“…Additionally, potentially less stable or more dynamic nucleoprotein filaments may be more effective at overcoming barriers, migrating joint molecules, and thereby completing the DNA strand exchange reaction. RAD51 nucleoprotein filaments were previously shown to grow from heterogeneous nuclei ranging in size from dimers, and even monomers, to large oligomers (54). Using smTIRFM, we were able to follow the formation kinetics of these nuclei at a singlemonomer resolution.…”

Spontaneous self-segregation of Rad51 and Dmc1 DNA recombinases within mixed recombinase filaments

“…Rad51/RecA family recombinases assemble through a well-defined pathway involving a rate limiting nucleation step, followed by more rapid spreading along the DNA (45)(46)(47)(48)(49)(50)(51)(52)(53). Within the context of this model, we can consider two possible mechanisms that might led to segregated Rad51 and Dmc1 filaments ( Figure 6A,B).…”