RNF8 ubiquitinates RecQL4 and promotes its dissociation from DNA double strand breaks

Qunsong Tan,Di Wei,Hongbo Fang,Yongliang Zhao,Yuqi Zhu,Mengge Li,Kaifeng Niu,Yueyang Li,Zixiang Chen,Adayabalam S Balajee

doi:10.1038/s41389-021-00315-0

Abstract

Ubiquitination-dependent DNA damage response (DDR) signals play a critical role in the cellular choice of DNA damage repair pathways. Human DNA helicase RecQL4 participates in DNA replication and repair, and loss of RecQL4 is associated with autosomal recessive genetic disorders characterized by genomic instability features. In an earlier study, RecQL4 was isolated as a stable complex that contained two ubiquitin ligases of the N-end rule (UBR1 and UBR2). However, it is unknown whether or not RecQL4 ubiquitination status is critical for its DNA repair function. Here, we report that RecQL4 directly interacts with RNF8 (a RING finger ubiquitin E3 ligase), and both co-localize at DNA double-strand break (DSB) sites. Our findings indicate that RNF8 ubiquitinates RecQL4 protein mainly at the lysine sites of 876, 1048, and 1101, thereby facilitating the dissociation of RecQL4 from DSB sites. RecQL4 mutant at ubiquitination sites had a significantly prolonged retention at DSBs, which hinders the recruitment of its direct downstream DSB repair proteins (CtIP & Ku80). Interestingly, reduced DSB repair capacity observed in RecQL4 depleted cells was restored only by the reconstitution of wild-type RecQL4, but not the ubiquitination mutant. Additionally, RecQL4 directly interacts with WRAP53β that is known to recruit RNF8 to DSBs and WRAP53β enhances the association of RecQL4 with RNF8. WRAP53β silencing resulted in a nearly diminished recruitment of RNF8 to DSBs and in a greatly attenuated dissociation of RecQL4 from the DSB sites. Collectively, our study demonstrates that the ubiquitination event mediated by RNF8 constitutes an essential component for RecQL4’s function in DSB repair.

Highlights

Posttranslational modifications of proteins, including nondegrading ubiquitination, precisely control the assembly or disassembly of DNA damage repair (DDR) proteins at DNA damage sites[1,2]
To search for other potential ubiquitin E3 ligase(s) that can modulate RecQL4 activity, we have screened a number of E3 ligases that have been reported to participate in the repair of DNA damage[16], and identified RNF8 as an interaction partner of RecQL4
Ubiquitination plays a pervasive role in the dynamic assembly and disassembly of DDR proteins on the DNA damage sites, where the E3 ligase RNF8 plays an essential role in initiating a cascade of ubiquitination in response to DNA double-strand breaks

Summary

Introduction

Posttranslational modifications of proteins, including nondegrading ubiquitination, precisely control the assembly or disassembly of DNA damage repair (DDR) proteins at DNA damage sites[1,2]. Ubiquitination, a covalent attachment of highly conserved single ubiquitin or complex polyubiquitin chains to lysine sites of proteins, provides quality controls through mediating DNA damage repair activities, and has been demonstrated to be a critical modification for the cellular choice of DNA repair pathways[3,4]. NHEJ pathway protein Ku80 is ubiquitinated by these two E3 ligases, and removed from chromatin in a valosin-containing protein (VCP)/p97dependent manner[9]. All these findings suggest a critical role of ubiquitination modification in modulating the activity of DNA damage repair proteins

Methods

Results

Conclusion