Abstract
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.
Highlights
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)
double-strand breaks (DSBs) are induced by ionizing irradiation (IR) or frequently arise during replication when forks collide with persistent single-strand breaks, such as those generated by camptothecin (CPT), a DNA topoisomerase I inhibitor[3]
We find that Kelch-like protein 15 (KLHL15) recognizes a short-tripeptide motif (FRY) located in the conserved C-terminal domain (CTD) of CtIP and that mutation of this motif protects CtIP from KLHL15dependent degradation resulting in prolonged CtIP protein half-life and, excessive DNA-end resection
Summary
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). Cells have evolved two major pathways dealing with the repair of DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR)[4]. DNA-end resection is initiated by the combined action of the MRE11–RAD50–NBS1 (MRN) complex and CtIP8, and is a key determinant of DSB repair pathway choice, as it commits cells to HR by preventing NHEJ9. We identify the human Kelch-like protein 15 (KLHL15), a substrate-specific adaptor for Cullin[3] (CUL3)-based E3 ubiquitin ligases, as a novel CtIP interaction partner. Our results uncover a critical role for CUL3-KLHL15 ubiquitin ligase in governing CtIP DNA-end resection activity and DSB repair pathway choice
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