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Non-Canonical Regulation of Homologous Recombination DNA Repair by the USP9X Deubiquitylase Rachel O’Dea and Corrado Santocanale*

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Non-Canonical Regulation of Homologous Recombination DNA Repair by the USP9X Deubiquitylase Rachel O’Dea and Corrado Santocanale* © 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs233437. doi:10.1242/jcs.233437 RESEARCH ARTICLE Non-canonical regulation of homologous recombination DNA repair by the USP9X deubiquitylase Rachel O’Dea and Corrado Santocanale* ABSTRACT mutations and it is therefore defined as an error-prone pathway. HR In order to prevent the deleterious effects of genotoxic agents, cells relies on DNA synthesis, using a homologous DNA sequence as the have developed complex surveillance mechanisms and DNA repair template to repair the break and is therefore a high-fidelity method pathways that allow them to maintain genome integrity. The ubiquitin- of repair (Sancar et al., 2004). specific protease 9X (USP9X) contributes to genome stability during The HR repair pathway is a complex hierarchical pathway in which DNA replication and chromosome segregation. Depletion of USP9X numerous proteins and post-translational modifications (PTMs) are leads to DNA double-strand breaks, some of which are triggered by critical. A key step in initiating HR repair after DSB recognition is the replication fork collapse. Here, we identify USP9X as a novel regulator recruitment of the E3 ligase BRCA1 and the nuclease CtIP (also of homologous recombination (HR) DNA repair in human cells. By known as RBBP8). This complex, together with additional nucleases performing cellular HR reporter, irradiation-induced focus formation resect the DNA DSB, revealing substantial single-stranded (ss)DNA and colony formation assays, we show that USP9X is required for committing the break to HR repair (Symington and Gautier, 2011). efficient HR. Mechanistically, we show USP9X is important to sustain The revealed ssDNA is coated initially with the ssDNA-binding RPA the expression levels of key HR factors, namely BRCA1 and RAD51 heterotrimer. At later stages in the pathway, RPA is replaced by the through a non-canonical regulation of their mRNA abundance. recombinase RAD51 (Krejci et al., 2012; Sugiyama et al., 1997). The Intriguingly, we find that the contribution of USP9X to BRCA1 and replacement of RPA with RAD51 is facilitated by BRCA1 through – RAD51 expression is independent of its known catalytic activity. Thus, recruitment of a PALB2 BRCA2 complex, which binds and loads this work identifies USP9X as a regulator of HR, demonstrates a novel RAD51 on to the ssDNA (Davies et al., 2017; Zhang et al., 2009). mechanism by which USP9X can regulate protein levels, and provides The RAD51 nucleofilament carries out the strand invasion and insights in to the regulation of BRCA1 and RAD51 mRNA. homology search required for DNA synthesis and the repair of the DSB (Shinohara et al., 1992). This article has an associated First Person interview with the first Protein ubiquitylation is one of the critical PTMs involved in HR. author of the paper. This PTM involves the addition of ubiquitin, an 8 kDa protein, to substrates via a three step enzymatic cascade consisting of a KEY WORDS: USP9X, DNA repair, BRCA1, RAD51, Deubiquitylation, ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme Homologous recombination (E2) and a ubiquitin ligase enzyme (E3). Substrates can be modified with a single moiety (monoubiquitylation) or polyubiquitin chains INTRODUCTION formed through a linkage using one of the seven lysine residues Cells are constantly exposed to agents that can damage our DNA. (K48, K63, K11, K27, K33, K6 or K29) or the N-terminal These can be from internal sources, such as products of metabolism methionine (M1) present in ubiquitin (Komander and Rape, 2012). and errors in DNA duplication, or from external sources, such as The presence of ubiquitin can affect substrate localisation, function carcinogens and UV light (Jackson and Bartek, 2009). DNA and stability (Akutsu et al., 2016). damage left unresolved or inaccurately repaired results in genome Several types of ubiquitin modifications are required to accomplish instability, leading to mutations, chromosome breakages and HR repair of DSB. The canonical K48 degradation ubiquitin signals translocations, events that can promote tumorigenesis (Khanna have been shown to regulate the stability of key HR proteins CtIP, and Jackson, 2001). To counteract this constitutive DNA damage, BRCA1 and the E3 ligase RNF168 (Gudjonsson et al., 2012; cells have intricate mechanisms in place for surveillance and repair Lafranchi et al., 2014; Lu et al., 2012; Wu et al., 2010). In addition, of DNA damage. DNA double-strand breaks (DSBs) are the most the K63 ubiquitin chains placed on histones by RNF8 and RNF168 deleterious type of DNA damage and two main mechanisms exist have been shown to be critical in generating a recruitment platform to repair this type of lesion: non-homologous end joining (NHEJ) for various mediator proteins (Doil et al., 2009). Non-canonical roles and homologous recombination (HR). NHEJ is an homology- of ubiquitin in HR have also been identified. The E3 complex, independent method of repair which relies on KU, DNA-PK and CRL3–KEAP1 has been shown to ubiquitylate PALB2 blocking its LIG IV to carry out minimal processing and ligation of broken DNA interaction with BRCA1 negatively regulating HR (Orthwein et al., ends (Chiruvella et al., 2013). Such processes very often introduce 2015). In addition, monoubiquitylation of histones has been shown to indirectly facilitate chromatin remodelling enabling the recruitment of HR proteins (Nakamura et al., 2011). Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway H91W2TY, Ireland. Protein ubiquitylation is a dynamic process and can be either fine- tuned or reversed by counteracting deubiquitylases (DUBs). The *Author for correspondence ([email protected]) negative regulation of ubiquitylation by DUBs has been shown to be R.O., 0000-0003-0487-5392; C.S., 0000-0003-1337-5656 critical for proper HR progression, as well as termination of the response to the DSB. The DUBs OTUB2, POH1 (also known as Received 18 April 2019; Accepted 30 December 2019 PSMD14) and BRCC36 (also known as BRCC3) have been shown Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs233437. doi:10.1242/jcs.233437 to directly antagonise histone RNF8 and RNF168 K63 chains their mRNA abundance. These findings offer novel insights into the (Butler et al., 2012; Kato et al., 2014; Shao et al., 2009), while regulation of BRCA1 and RAD51, demonstrate a novel role for USP3, USP16 and USP44 have been shown to remove histone USP9X in DNA repair, and potentially provide a novel mechanistic monoubiquitin moieties (Mosbech et al., 2013; Nicassio et al., explanation for the tumour-suppressive properties of USP9X. 2007; Shanbhag et al., 2010). This DUB activity is critical in preventing excessive modifications and removing the signal after RESULTS the break has been repaired. There is also evidence of non-canonical Loss of USP9X leads to accumulation of DSBs DUB functions in HR. OTUB1 has been shown to regulate HR One of the earliest events in the response to DNA DSBs is the independent of its catalytic activity, through inhibitory binding of phosphorylation of the histone variant H2AX (encoded by H2AFX) RNF168 E2 ligase, UBC13 (Nakada et al., 2010). on serine 139 on the chromatin surrounding the break. This Ubiquitin-specific protease 9X (USP9X) is a member of the USP modification, also known as γ-H2AX, is removed upon completion family of DUBs, a large family of DUBs that typically lack of DSB repair (Chowdhury et al., 2005), thus γ-H2AX is a widely specificity for particular UB chains types (Faesen et al., 2011). accepted as a marker of DSBs. In previous work, we have reported USP9X in a cellular context has been shown to have the ability to that in the human embryonic kidney derived HEK-293 cells, remove K48, K63, K33 and K29 ubiquitin chains as well as USP9X depletion leads to an increase the number of γ-H2AX- monoubiquitin (Al-Hakim et al., 2008; Dupont et al., 2009; Marx positive cells (McGarry et al., 2016). In order to extend these et al., 2010; Mouchantaf et al., 2006; Vong et al., 2005). observations, we used a different cell line and additional siRNA Predominantly by promoting K48 chain hydrolysis, USP9X has sequences. Osteosarcoma-derived U2OS cells were transfected with been implicated in a variety of different cellular events. Originally two different non-overlapping siRNAs [denoted siUSP9X (a) and discovered for its role in deciding cell fate decisions during siUSP9X (b)], and the number of γ-H2AX foci detectable after 48 h development in mice (Fischer-Vize et al., 1992), USP9X is now was quantified using high-throughput microscopy. Approximately known to regulate the levels of proteins implicated in a variety of 300 nuclei in three independent experiments were scored. We different cellular processes, including DNA replication, centrosome observed that the average number of γ-H2AX foci was increased biogenesis and signalling pathways (Dupont et al., 2009; Izrailit when USP9X was depleted with both of the siRNAs (Fig. 1A,B). et al., 2016; Li et al., 2017; McGarry et al., 2016; Wang et al., 2017; Decreased levels of USP9X upon siRNA transfections were Wu et al., 2017). confirmed by western blot analysis (Fig. 1C), suggesting that the USP9X is deregulated in a number of different cancers and has depletion of USP9X leads to the accumulation of DSB. been shown to act as both a tumour suppressor and an oncogene, The accumulation of DNA damage in USP9X-depleted depending on the type and stage of cancer (Cox et al., 2014). proliferating cells may simply be due to the role of USP9X in Overexpression of USP9X has been identified in cervical, protecting claspin levels and replication forks from collapsing colorectal, kidney, breast, prostate, brain and sarcoma cancers during S-phase (McGarry et al., 2016); however, we showed that the (Murtaza et al., 2015). The oncogenic function of USP9X was overexpression of claspin in USP9X-depleted cells did not fully initially explained by its ability to stabilise the anti-apoptotic factor prevent the accumulation of DNA damage, suggesting there are MCL1.
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