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NUCKS1 Promotes RAD54 Activity in Homologous Recombination DNA Repair

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NUCKS1 Promotes RAD54 Activity in Homologous Recombination DNA Repair ARTICLE NUCKS1 promotes RAD54 activity in homologous recombination DNA repair David G. Maranon1*, Neelam Sharma1*, Yuxin Huang3*, Platon Selemenakis1,2, Meiling Wang3, Noelia Altina1,2,WeixingZhao3,and Claudia Wiese1,2 NUCKS1 (nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate 1) is a chromatin-associated, vertebrate- specific, and multifunctional protein with a role in DNA damage signaling and repair. Previously, we have shown that NUCKS1 helps maintain homologous recombination (HR) DNA repair in human cells and functions as a tumor suppressor in mice. However, the mechanisms by which NUCKS1 positively impacts these processes had remained unclear. Here, we show that NUCKS1 physically and functionally interacts with the DNA motor protein RAD54. Upon exposure of human cells to DNA- damaging agents, NUCKS1 controls the resolution of RAD54 foci. In unperturbed cells, NUCKS1 prevents RAD54’s inappropriate engagement with RAD51AP1. In vitro, NUCKS1 stimulates the ATPase activity of RAD54 and the RAD51–RAD54- mediated strand invasion step during displacement loop formation. Taken together, our data demonstrate that the NUCKS1 protein is an important new regulator of the spatiotemporal events in HR. Introduction Homologous recombination (HR) DNA repair is a complex DNA Nuclear ubiquitous casein kinase and cyclin-dependent ki- damage repair pathway that serves to maintain genome stability nase substrate 1 (NUCKS1) is a nuclear, ubiquitously expressed, and suppress cancer. HR is intimately linked to DNA replication, chromatin-associated, and highly posttranslationally modified and the involvement of HR in the recovery of stalled and col- protein (Grundt et al., 2004; Grundt et al., 2007; Grundt et al., lapsed replication forks ensures the faithful replication and 2002; Parplys et al., 2015; Wisniewski et al., 2008). NUCKS1 segregation of chromosomes (Lambert et al., 2007). HR entails shares extensive amino acid sequence homology with RAD51- template-dependent DNA strand invasion and DNA synthesis to associated protein 1 (RAD51AP1; Parplys et al., 2015), a RAD51- repair or tolerate DNA damage, such as DNA double-strand binding protein intimately involved in HR DNA repair (Dray breaks (DSBs), single-stranded DNA (ssDNA) gaps, and inter- et al., 2011; Dray et al., 2010; Dunlop et al., 2012; Dunlop et al., strand DNA cross-links (Daley et al., 2014; Daley et al., 2013; Li 2011; Modesti et al., 2007; Wiese et al., 2007). Based on this and Heyer, 2008). In the HR reaction, the 59 strand at the DNA finding, we previously tested and uncovered a role for NUCKS1 break site undergoes a resection to generate a 39 ssDNA over- in HR DNA repair and DNA replication (Parplys et al., 2015). In hang (Daley et al., 2015; Symington, 2014). This overhang experiments involving gene-specific knockdown of NUCKS1 by is quickly protected by the ssDNA-binding protein RPA. For the siRNA, we showed that the down-regulation of NUCKS1 impairs HR reaction to continue, RPA must be replaced by the RAD51 HR in human cells. We also showed that loss of NUCKS1 function recombinase, a rate-limiting process that is dependent on the renders S/G2-phase cells hypersensitive to ionizing radiation action of multiple HR mediators (Dosanjh et al., 1998; Godin (IR) and that NUCKS1 loss increases cellular sensitivity to che- et al., 2016; Sung, 1997; Sung et al., 2003; Zelensky et al., 2014; motherapeutic agents and the susceptibility to IR-induced can- Zhao et al., 2015). Then, the RAD51-ssDNA nucleoprotein fila- cers in mice (Parplys et al., 2015; Yue et al., 2016). However, the ment, also known as the presynaptic filament, captures the mechanisms by which NUCKS1 influences HR and confers tumor duplex DNA and generates a displacement loop (D-loop) upon suppression had remained unclear. location of the homologous DNA target sequence. Subsequent The DNA motor protein RAD54 belongs to the SWI2/SNF2 DNA synthesis and resolution completes the HR reaction (Daley protein family of DNA-dependent ATPases and has multiple et al., 2013). functions in HR-mediated DSB repair (Ceballos and Heyer, 2011). ............................................................................................................................................................................. 1Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO; 2Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO; 3Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX. *D.G. Maranon, N. Sharma, and Y. Huang contributed equally to this paper; Correspondence to Claudia Wiese: [email protected]; Weixing Zhao: [email protected]. © 2020 Maranon et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). Rockefeller University Press https://doi.org/10.1083/jcb.201911049 1of15 J. Cell Biol. 2020 Vol. 219 No. 10 e201911049 One critical function of RAD54 lies after presynaptic filament To explore if NUCKS1 status affects RAD54 foci formation, formation. Upon the successful engagement of a homologous exponentially growing cell cultures were exposed to 8 Gy duplex sequence by the RAD51-ssDNA filament, RAD54 pro- γ-irradiation and then fixed and stained for RAD54 at 4 h, 8 h, motes the conversion of the synaptic complex into a heterodu- and 24 h after exposure. RAD54 foci were more prevalent in plex DNA joint, the D-loop (Jaskelioff et al., 2003; Petukhova NUCKS1 KO than in control cells at all times after IR exposure et al., 1999; Solinger and Heyer, 2001; Solinger et al., 2001; (Fig. 1 D and Fig. S1 D). Importantly, for all cell lines investigated, Van Komen et al., 2002). RAD54 also removes RAD51 from the the expression levels of RAD54 protein and cell cycle profiles nascent D-loop, so as to make the 39 end in the D-loop accessible were similar (Fig. 1 C and Fig. S2, A and B). NUCKS1 KO cells also to DNA polymerases for repair synthesis (Ceballos and Heyer, contained more RAD54 foci upon exposure of cells to the in- 2011; Liu et al., 2017; Wright and Heyer, 2014). These functions terstrand cross-linking agent mitomycin C (MMC; Fig. 1 E and of RAD54 require its ATPase activity. Independent of its ATPase Fig. S2 E), and, after MMC treatment, the cell cycle profiles for activity, RAD54 stabilizes the RAD51 nucleoprotein filament and NUCKS1-deficient cells were not different from control cells promotes the robustness of DNA damage–induced RAD51 foci in (Fig. S2 F). Importantly, at 24 h after treatment of cells with cells (Agarwal et al., 2011; Mazin et al., 2003; Tanetal.,1999; van either IR or MMC, significantly more nuclei with RAD54 foci Veelen et al., 2005). remained in NUCKS1 KO cells compared with control cells (Fig. 1, In this study, we show that the NUCKS1 protein is an im- DandE). To exclude cell type–specific effects, we used CRISPR/ portant new regulator of the spatiotemporal events in the HR Cas9 to generate NUCKS1-deficient HT1080 cells (Fig. S2 C). reaction. Upon exposure of human cells to DNA-damaging Upon exposure of these cells to γ-irradiation, more nuclei with agents, NUCKS1 is required for the timely resolution of DNA RAD54 foci were detected in NUCKS1-deficient HT1080 cells damage–induced RAD54 foci. NUCKS1 is epistatic with RAD54, compared with control cells (Fig. S2 D). Together, these results interacts with RAD54 in vitro and in cells, and functions in show that DNA damage–induced RAD54 foci are more abundant conjunction with RAD54 in the RAD51-mediated DNA joint and persist longer in NUCKS1-deficient cells. molecule formation as measured by the D-loop assay. NUCKS1 Next, we tested if knockdown of RAD54 in NUCKS1 KO cells competes with RAD51AP1 to interact with RAD54. As such, would further increase their sensitivity to MMC. As observed NUCKS1 spatially and temporally controls the progression of the for HeLa cells depleted for NUCKS1 (Parplys et al., 2015), NUCKS1 HR pathway. Consistent with a role for NUCKS1 in genome KO HeLa cells showed increased sensitivity to the cytotoxic ef- stability and tumor suppression, HR impairment in NUCKS1- fects of MMC (Fig. S2 G). NUCKS1 KO cells transfected with a deficient cells leads to the increased utilization of error-prone nondepleting siRNA, and NUCKS1 KO cells and HeLa cells each nonhomologous end joining (NHEJ) to facilitate DSB repair. depleted for RAD54 showed the same sensitivity to the cytotoxic effects of MMC (Fig. 1, F and G). These results suggest epistasis between NUCKS1 and RAD54 in human cells after MMC Results treatment. NUCKS1 is required for the timely resolution of DNA damage–induced RAD54 foci NUCKS1 deficiency delays the formation of DNA Human NUCKS1 is a 243–amino acid residue protein containing damage–induced RAD51 foci two peptide motifs for nuclear localization (NLS1 and NLS2) and We endeavored to test if RAD54 function was compromised in one DNA-binding domain (Fig. 1 A; Grundt et al., 2007; Grundt NUCKS1 KO cells. Since defects in RAD54 were shown to di- et al., 2002). Human cells depleted for NUCKS1 (by siRNA) are minish the robustness of RAD51 foci (Agarwal et al., 2011; Tan impaired in HR and sensitive to DNA damage–inducing agents et al., 1999; van Veelen et al., 2005), we tested if this also was the but can form damage-induced RAD51 foci (Parplys et al., 2015). case in NUCKS1 KO cells. Compared with HeLa cells, less RAD51 Similar to RAD51, the DNA motor protein RAD54 also accu- foci were detected in NUCKS1 KO cells at early times (2 h and 4 h) mulates into dynamic protein foci at the sites of DNA damage after IR exposure (Fig. 2 A and Fig.
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