The Eukaryotic homology search complex distorts donor DNA structure to probe for homology.

Homologous recombination (HR) is a DNA double-strand break repair pathway that facilitates genetic exchange and protects damaged replication forks during DNA synthesis. As a template-based repair process, the successful repair of a double-strand break depends on locating suitable homology from a donor DNA sequence elsewhere in the genome. In eukaryotes, Rad51 catalyzes the homology search in coordination with the ATP-dependent motor protein Rad54. The mechanism by which these two proteins regulate forces on dsDNA substrates during homology search remains unknown. Here, we have utilized single-molecule magnetic tweezers and optical trapping methods to monitor remodeling of the DNA template during the homology search. We find that the activity of Rad51 and Rad54 remodels the donor DNA substrate to control the association and dissociation of Rad51-ssDNA filaments in the absence of DNA homology. This mechanism occurs through the application of both linear (tension) and rotational (torsion) forces on the donor DNA. Finally, failure of Rad54 to act processively disrupts target selection in vivo . This study provides a basic understanding of how motorized homology search manipulates the donor DNA during the search for a suitable repair template. SIGNIFICANCE STATEMENT: Homologous recombination (HR) is a double-strand DNA break repair pathway that utilizes a template-based target search process to locate a suitable homologous DNA sequence in the genome, thereby initiating DNA repair. Called the homology search, in eukaryotes, this process is carried out by the RecA family member Rad51. During the homology search, Rad51 collaborates with the motor protein Rad54 to identify and interrogate homologous DNA sequences within the genome. In this study, we have measured the forces applied by the combination of Rad51 and Rad54 to the donor DNA duplex. These measurements reveal a coordinated effort by these motor proteins to remodel donor DNA to probe for homology, shedding new light on how template-based homology searches interrogate the DNA strands.