Search

Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we directly observe the widely debated mechanism of RecA homology searching at a single-molecule level using high-speed atomic force microscopy (HS-AFM) in combination with tailored DNA origami frames to present the reaction targets in a way suitable for AFM-imaging. We show that RecA nucleoprotein filaments move along DNA substrates via short-distance facilitated diffusions, or slides, interspersed with longer-distance random moves, or hops. Importantly, from the specific interaction geometry, we find that the double-stranded substrate DNA resides in the secondary DNA binding-site within the RecA nucleoprotein filament helical groove during the homology search. This work demonstrates that tailored DNA origami, in conjunction with HS-AFM, can be employed to reveal directly conformational and geometrical information on dynamic protein-DNA interactions which was previously inaccessible at an individual single-molecule level.

Protein-mediated self-assembly is arguably one of the most promising routes for building complex molecular nanostructures. Here, we report a molecular self-assembly technique that allows programmable, site-specific patterning of double-stranded DNA scaffolds, at a single-base resolution, by 3-nm-long RecA-based nucleoprotein filaments. RecA proteins bind to single-stranded DNA to form nucleoprotein filaments. These can self-assemble onto a double-stranded DNA scaffold at a region homologous to the nucleoprotein's single-stranded DNA sequence. We demonstrate that nucleoprotein filaments can be formed from single-stranded DNA molecules ranging in length from 60 nucleotides down to just 6 nucleotides, and these can be assembled site-specifically onto a model DNA scaffold both at the end of the scaffold and away from the end. In both cases, successful site-specific self-assembly is demonstrated even for the smallest nucleoprotein filaments, which are just 3 nm long, comprise only two monomers of RecA, and cover less than one helical turn of the double-stranded DNA scaffold. Finally, we demonstrate that the RecA-mediated assembly process is highly site-specific and that the filaments indeed bind only to the homologous region of the DNA scaffold, leaving the neighboring scaffold exposed.