Abstract
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.
Highlights
The repair of DNA damage, in particular double stranded breakages, is critical to the maintenance of genomic integrity in all forms of life
recombinase A (RecA) nucleoprotein filaments were imaged in situ with HSAFM while searching for homology on the double-stranded DNA (dsDNA) presented by the DNA origami nanostructures
We have demonstrated that high-speed atomic force microscopy (HS-atomic force microscopy (AFM)) combined with the DNA origami frame system, where a biological interaction can take place within a structurally robust reference, can provide conformational and geometrical information on dynamic nucleoprotein interactions at a single-molecule level, revealing details unobtainable by any other method
Summary
The repair of DNA damage, in particular double stranded breakages, is critical to the maintenance of genomic integrity in all forms of life. Homologous recombination is mediated by the ubiquitous recombinase A (RecA) family of recombinases and involves three steps: formation of a nucleoprotein filament; location of sequence homology; and strand exchange. Once formed, the nucleoprotein complex searches available and accessible double-stranded DNA (dsDNA) for sequence homology between the encapsulated ssDNA and the dsDNA substrate.[2] Molecular dynamics (MD) simulations suggest that the incoming dsDNA enters the filament via the filament’s helical groove,[3] giving rise to a characteristic interaction geometry which is as yet unverified experimentally. Binding occurs via salt bridges formed with the backbone of the strand that is not probed for homology, the outgoing strand, leaving the other complementary strand of the dsDNA free to interact with the encapsulated ssDNA.[3]
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