Multifaceted roles of H2B mono-ubiquitylation in D-loop metabolism during homologous recombination repair

SUMMARY Repairing DNA double-strand breaks is crucial for maintaining genome integrity, which occurs primarily through homologous recombination (HR) in S. cerevisiae. Nucleosomes, composed of DNA wrapped around a histone octamer, present a natural barrier to end-resection to initiate HR, but the impact on the downstream HR steps of homology search, DNA strand invasion and repair synthesis remain to be determined. Displacement loops (D-loops) play a pivotal role in HR, yet the influence of chromatin dynamics on D-loop metabolism remains unclear. Using the physical D-loop capture (DLC) and D-loop extension (DLE) assays to track HR intermediates, we employed genetic analysis to reveal that H2B mono-ubiquitylation (H2Bubi) affects multiple steps during HR repair. We infer that H2Bubi modulates chromatin structure, not only promoting histone degradation for nascent D-loop formation but also stabilizing extended D-loops through nucleosome assembly. Furthermore, H2Bubi regulates DNA resection via Rad9 recruitment to suppress a feedback control mechanism that dampens D-loop formation and extension at hyper-resected ends. Through physical and genetic assays to determine repair outcomes, we demonstrate that H2Bubi plays a crucial role in preventing break-induced replication and thus promoting genomic stability. Highlights H2Bubi is epistatic to H2A.Z and INO80 in promoting homology search and D-loop formation H2Bubi stabilizes extended D-loop Excessive resection counteracts D-loop formation and extension H2Bubi promotes crossover events and limits the frequency of break-induced replication outcomes in HR repair Full Text Availability The license terms selected by the author(s) for this preprint version do not permit archiving in PMC. The full text is available from the preprint server.