Highlights d CtIP protects reversed forks from nucleolytic degradation d CtIP prevents DNA2-dependent over-resection of nascent strands d CtIP nuclease mutants are defective in fork protection d CtIP-mediated fork protection synergizes with BRCA1, not BRCA2

Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein–protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.

DNA double-strand breaks (DSBs) are one of the most detrimental lesions, as their incorrect or incomplete repair can lead to genomic instability, a hallmark of cancer. Cells have evolved two major competing DSB repair mechanisms: Homologous recombination (HR) and non-homologous end joining (NHEJ). HR is initiated by DNA-end resection, an evolutionarily conserved process that generates stretches of single-stranded DNA tails that are no longer substrates for religation by the NHEJ machinery. Ubiquitylation and sumoylation, the covalent attachment of ubiquitin and SUMO moieties to target proteins, play multifaceted roles in DNA damage signaling and have been shown to regulate HR and NHEJ, thus ensuring appropriate DSB repair. Here, we give a comprehensive overview about the current knowledge of how ubiquitylation and sumoylation control DSB repair by modulating the DNA-end resection machinery.