Sequential adaptor function of Treacle and MDC1 couples nucleolar reorganization to RNF8-dependent recruitment of HDR factors

Abstract

The repair of DNA double-strand breaks in repetitive sequences is challenging because the abundance of potential templates for homology-directed repair (HDR) increases the risk of ectopic recombination and chromosome rearrangements. Relocalization of repair sites in repetitive sequences to a safe location prior to RAD51 loading has been observed in various organisms and is thought to limit aberrant recombination events. Here, we characterize this process in ribosomal DNA (rDNA) repeats, which reside within the nucleoli, specialized nuclear compartments dedicated to ribosome biogenesis. Induction of DSBs in rDNA triggers large-scale mobilization of damaged repeats from the nucleolar interior to the nucleolar periphery, where repair occurs. We show that this process is coordinated by the adaptor proteins Treacle and MDC1, which act sequentially to control nucleolar reorganization and repair factor recruitment. Treacle promotes rDNA mobilization through its role in nucleolar DNA damage signalling, whereas MDC1 acts downstream of nucleolar segregation to facilitate the recruitment of HDR factors at nucleolar caps. Following establishment of a {gamma}H2AX chromatin domain, MDC1-dependent RNF8-mediated chromatin ubiquitylation promotes BRCA1 recruitment via both the RNF168-dependent pathway and the RAP80-ABRAXAS complex. This in turn enables the sequential accumulation of PALB2 and RAD51 at nucleolar caps. Together, our findings demonstrate that RAD51 loading at rDNA breaks is tightly coupled to nucleolar reorganization and is mediated by dual RNF8-dependent signalling pathways, thereby ensuring that HDR is spatially restricted to the nucleolar periphery and reducing the risk of ectopic recombination between repetitive sequences.