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 single-molecule 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.
Highlights
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
A Double-stranded DNA (dsDNA) molecule (48.5 or 38.4 kbp) labeled with biotins at the 3′ and 5′ ends of the same strand was captured from both ends with two streptavidincoated microbeads held by independent dual-trap optical tweezers
The single-stranded DNA (ssDNA) molecule was repositioned in the imaging channel and inspected using fluorescence microscopy (Fig. 1 A and B)
Summary
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. The ATP-dependent recombinase protein RAD51, the focus of this study, must 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. The mechanism of RAD51-recombinase filament formation is visualized and quantified with single-molecule resolution using a combination of dual optical tweezers, fluorescence microscopy, and microfluidics With this method, short-lived transient intermediates formed during nascent RAD51 filament assembly were observed directly. Both magnetic-tweezers studies, were performed at low salt concentrations, conditions known to favor dsDNA binding Such observations show that the RAD51 filament formation mechanism is complex and may be affected by many parameters [16]. Obtaining further insight into the RAD51 filament formation mechanism would benefit from direct observation and quantification of nucleation and growth, separately and in the absence of disassembly, under identical experimental conditions on ssDNA and dsDNA with the same sequence
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