The de-ubiquitylating enzymes USP26 and USP37 regulate homologous recombination by counteracting RAP80.

Dimitris Typas,Leon H Mullenders,Michaela Diakatou,Bram Van De Broek,Haico Van Attikum,Peter E Thijssen,Wouter W Wiegant,Martijn S Luijsterburg,Angela Helfricht

doi:10.1093/nar/gkv613

Abstract

The faithful repair of DNA double-strand breaks (DSBs) is essential to safeguard genome stability. DSBs elicit a signaling cascade involving the E3 ubiquitin ligases RNF8/RNF168 and the ubiquitin-dependent assembly of the BRCA1-Abraxas-RAP80-MERIT40 complex. The association of BRCA1 with ubiquitin conjugates through RAP80 is known to be inhibitory to DSB repair by homologous recombination (HR). However, the precise regulation of this mechanism remains poorly understood. Through genetic screens we identified USP26 and USP37 as key de-ubiquitylating enzymes (DUBs) that limit the repressive impact of RNF8/RNF168 on HR. Both DUBs are recruited to DSBs where they actively remove RNF168-induced ubiquitin conjugates. Depletion of USP26 or USP37 disrupts the execution of HR and this effect is alleviated by the simultaneous depletion of RAP80. We demonstrate that USP26 and USP37 prevent excessive spreading of RAP80-BRCA1 from DSBs. On the other hand, we also found that USP26 and USP37 promote the efficient association of BRCA1 with PALB2. This suggests that these DUBs limit the ubiquitin-dependent sequestration of BRCA1 via the BRCA1-Abraxas-RAP80-MERIT40 complex, while promoting complex formation and cooperation of BRCA1 with PALB2-BRCA2-RAD51 during HR. These findings reveal a novel ubiquitin-dependent mechanism that regulates distinct BRCA1-containing complexes for efficient repair of DSBs by HR.

Highlights

DNA double-strand breaks (DSBs) pose a considerable threat to the stability of the human genome
While the BRCA1-PALB2-BRCA2-RAD51 (BRCC) complex promotes homologous recombination (HR), the BRCA1-AbraxasRAP80-MERIT40 (BRCA1-A) complex functionally antagonizes this repair process either by limiting DNA endresection or sequestering BRCA1 away from HR sites by binding to RNF8/RNF168-ubiquitylated chromatin [16,35,36,37,38,39,40]. These findings suggest that distinct BRCA1containing complexes can differentially affect HR in a manner that depends on the activity of the RNF8/RNF168 ubiquitin response
Given that 53BP1 directly binds to RNF168-induced ubiquitin conjugates [9,11], we reasoned that de-ubiquitylating enzymes (DUBs) modulating both these processes are likely to regulate RNF168-mediated HR events

Summary

Introduction

DNA double-strand breaks (DSBs) pose a considerable threat to the stability of the human genome. Their timely repair is essential to safeguard genome stability, and counteracts tumor development [1]. The binding of the RNF8 E3 ubiquitin ligase to MDC1 subsequently initiates a ubiquitylation-dependent cascade, involving the recruitment of the E3 ubiquitin ligase RNF168 in cooperation with the E2 ubiquitin-conjugating enzyme UBC13 [7,8] The activity of these enzymes contributes to the ubiquitylation of K13/15 on histone H2A/H2AX [9,10], as well as the ubiquitin-dependent assembly of 53BP1 [11], RAD18 [12] and the BRCA1-Abraxas-RAP80-MERIT40 (or BRCA1A) complex [13,14,15,16] onto DSB-neighboring chromatin

Methods

Results

Conclusion