Highlights d Rad54 acts as a molecular motor that guides Rad51 ssDNA along a donor dsDNA d Rad54 opens the donor DNA, allowing the search take place within an underwound bubble d Homology recognition is dependent on RPA and also on strand polarity

In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3′ singlestrand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use singlemolecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1-dependent or -independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1-Dna2-Top3-Rmi1-RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.DNA repair | homologous recombination | single molecule | helicase | DNA end resection

Ngo et al. use single-molecule methods to show that DNA can be more readily displaced from one side of a nucleosome relative to the other side. This unexpected mechanical asymmetry may offer a path of least resistance, allowing RNA polymerases to traverse nucleosomes if they approach from the correct direction.

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