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

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.

Role and Regulation of the RECQL4 Family during Genomic Integrity Maintenance

“…Cellular extracts from RECQL4 knockdown cells have decreased capacity to join DNA ends in vitro . This was recapitulated in vivo as RECQL4 knockdown leads to decreased NHEJ using a GFP reporter system and similar results were later confirmed by others [ 52 , 74 , 89 ]. Shamanna et al also demonstrated that RECQL4 physically and functionally interacts with the key NHEJ protein, KU70, in the N-terminus of RECQL4.…”

“…Furthermore, addition of RECQL4 increases KU complex DNA binding in vitro [ 52 ]. KU complex interaction with RECQL4 was later confirmed in vivo ( Table 2 ) [ 74 , 89 ]. Lu et al demonstrated that RECQL4 interacts with KU70 in a cell cycle dependent manner with increased interaction during the G1 stage, when NHEJ is the predominant DSB repair pathway ( Figure 3 C) [ 74 , 90 ].…”

“…RECQL4 has roles during the two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR) ( Figure 3 C; [ 52 , 53 , 74 , 89 ]. In 2014, Shamanna et al demonstrated that RECQL4 knockdown leads to decreased end joining both in in vitro and in vivo [ 52 ].…”

Molecular Mechanisms of the RECQ4 Pathogenic Mutations

“…Even though the residues required for RECQ4 recruitment to DNA damage sites have been identified ( Singh et al, 2010 ), it is yet to be determined if these residues are involved in direct DNA damage recognition or RECQ4 re-localization to the damage site via protein-protein interaction(s). Once bound to the DNA damage, RECQ4 requires RNF8-dependent ubiquitination at its C-terminus to dissociate from the site of DNA damage, allowing downstream DNA repair factors to bind to the damage site ( Tan et al, 2021 ). While studies show that RECQ4 positively regulates homologous recombination and non-homologous end joining, RECQ4 suppresses the repair of DNA breaks via RAD52-mediated single-strand annealing pathway via its SF2 and/or the C-terminal domains ( Kohzaki et al, 2020 ).…”

“…These enhanced activities were not observed in cells expressing the del(Ala420-Ala463) mutant protein, providing an explanation for why cells expressing Pro466Leu RECQ4 mutant do not contain high levels of mtDNA compared to those expressing the del(Ala420-Ala463) mutant ( Croteau et al, 2012b ; Wang et al, 2014 ; Chang et al, 2020 ). It would be a great interest to determine if the enhanced nucleic acid affinity by the Pro466Leu mutation may retain RECQ4 at DNA damage sites, hindering the binding and access of downstream DNA repair factors ( Tan et al, 2021 ). Nonetheless, another report showed that Pro466Leu, Phe637Ser and Phe697Leu disease mutations decrease RECQ4 DNA binding ( Jensen et al, 2012 ).…”