MutSβ protects common fragile sites by facilitating homology-directed repair at DNA double-strand breaks with secondary structures.

Common fragile sites (CFSs) are regions prone to chromosomal rearrangements, thereby contributing to tumorigenesis. Under replication stress (RS), CFSs often harbor under-replicated DNA regions at the onset of mitosis, triggering homology-directed repair known as mitotic DNA synthesis (MiDAS) to complete DNA replication. In this study, we identified an important role of DNA mismatch repair protein MutSβ (MSH2/MSH3) in facilitating MiDAS and maintaining CFS stability. Specifically, we demonstrated that MutSβ is required for the increased mitotic recombination induced by RS or FANCM loss at CFS-derived AT-rich and structure-prone sequences (CFS-ATs). We also found that MSH3 exhibits synthetic lethality with FANCM. Mechanistically, MutSβ is required for homologous recombination (HR) especially when DNA double-strand break (DSB) ends contain secondary structures. We also showed that upon RS, MutSβ is recruited to Flex1, a specific CFS-AT, in a PCNA-dependent but MUS81-independent manner. Furthermore, MutSβ interacts with RAD52 and promotes RAD52 recruitment to Flex1 following MUS81-dependent fork cleavage. RAD52, in turn, recruits XPF/ERCC1 to remove DNA secondary structures at DSB ends, enabling HR/break-induced replication (BIR) at CFS-ATs. We propose that the specific requirement of MutSβ in processing DNA secondary structures at CFS-ATs underlies its crucial role in promoting MiDAS and maintaining CFS integrity.