Rad54 separation of function mutation highlights unique roles during homologous recombination

Hu, J.; Moraga, D.; Xu, A.; Peysakhova, L.; Crickard, J. B.

doi:10.1101/2025.10.15.682645

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bioRxiv · 10.1101/2025.10.15.682645

Rad54 separation of function mutation highlights unique roles during homologous recombination

Hu, J.·Moraga, D.·Xu, A.·Peysakhova, L.·Crickard, J. B.

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Abstract

Homologous recombination (HR) is a DNA repair pathway that utilizes a template-based approach to repair double-strand breaks within the genome. Template utilization requires the exchange of individual DNA strands, which members of the RecA family of recombinases facilitate. Rad51 is a primary strand exchange factor in eukaryotes. During regular mitotic DNA repair, Rad51 is aided by the DNA translocase Rad54, which acts as a motor to remodel the template DNA and stabilize primary-strand exchange intermediates. The regulation of this activity remains incompletely understood. Here, we have identified a conserved site within the C-terminal region of Rad54. The mutation of this site creates a functional separation at early strand-exchange intermediates in vivo. Using this mutant protein, we identify a novel intermediate essential for stabilizing displacement loop (D-loop) structures. This precedes the removal of Rad51 and DNA extension. Based on our experiments, we hypothesize that this Rad54 mutant cannot stabilize Rad51-mediated strand-exchange intermediates because it cannot topologically isolate dsDNA regions. Identifying a mutant that disrupts this intermediate before Rad51 removal unifies existing models of Rad54-mediated D-loop formation and extension.

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Hu, J., Moraga, D., Xu, A., Peysakhova, L., Crickard, J. B.. 2025-10-15. Rad54 separation of function mutation highlights unique roles during homologous recombination. https://doi.org/10.1101/2025.10.15.682645

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