Structural Basis for Shieldin Complex Subunit 3–Mediated Recruitment Of

ARTICLE cro Structural basis for shieldin complex subunit 3–mediated recruitment of the checkpoint protein REV7 during DNA double-strand break repair Received for publication, October 12, 2019, and in revised form, November 27, 2019 Published, Papers in Press, December 3, 2019, DOI 10.1074/jbc.RA119.011464 Yaxin Dai‡§, Fan Zhang¶, Longge Wang‡§, Shan Shan‡, X Zihua Gong¶1, and X Zheng Zhou‡§2 From the ‡National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the §Institute of Biophysics, University of Chinese Academy of Sciences, Beijing 100049, China, and the ¶Department of Cancer Biology, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio 44195 Edited by Patrick Sung Shieldin complex subunit 3 (SHLD3) is the apical subunit of a properly repaired. In vertebrate cells, two main repair path- recently-identified shieldin complex and plays a critical role in ways, nonhomologous end-joining (NHEJ) and homologous DNA double-strand break repair. To fulfill its function in recombination (HR), are employed in eliminating the cytotoxic Downloaded from DNA repair, SHLD3 interacts with the mitotic spindle assembly DSBs and thereby ensuring genomic integrity (1, 2). The deci- checkpoint protein REV7 homolog (REV7), but the details of sion-making process of repair pathways is a critical step during this interaction remain obscure. Here, we present the crystal DSB response, which involves several elements such as the cell structures of REV7 in complex with SHLD3’s REV7-binding cycle state, the epigenetic regulation, and the DNA end resec- domain (RBD) at 2.2–2.3 Å resolutions. The structures revealed tion (3, 4). 53BP1, in concert with its upstream regulators and http://www.jbc.org/ that the ladle-shaped RBD in SHLD3 uses its N-terminal loop downstream effectors, spearheads the restriction of end resection and C-terminal ␣-helix (␣C-helix) in its interaction with REV7. to promote NHEJ and antagonizes BRCA1-mediated HR repair, The N-terminal loop exhibited a structure similar to those pre- whose initiation step relies on resected DNA termini (5–14). viously identified in other REV7-binding proteins, and the less- Recently, several elegant studies have identified a previously-un- conserved ␣C-helix region adopted a distinct mode for binding characterized protein complex, named Shieldin, working as a at Inst Biophys, CAS on February 23, 2020 REV7. In vitro and in vivo binding analyses revealed that the downstream effector of 53BP1 to protect DNA end from resection N-terminal loop and the ␣C-helix are both indispensable and to facilitate NHEJ repair in a 53BP1-dependent manner (15– for high-affinity REV7 binding (with low-nanomolar affinity), 21). Similar to loss of 53BP1, deficient Shieldin leads to reactiva- underscoring the crucial role of SHLD3 ␣C-helix in protein tion of end resection, impairs NHEJ activities, and affects the binding. Moreover, binding kinetics analyses revealed that the immunoglobulin class switching process (22). REV7 “safety belt” region, which plays a role in binding other Shieldin consists of four subunits: SHLD3 (also known as proteins, is essential for SHLD3–REV7 binding, as this region CTC-534A2.2 or RINN1); SHLD2 (also known as FAM35A or retards the dissociation of the RBD from the bound REV7. RINN2); SHLD1 (also known as C20ORF196 or RINN3); and Together, the findings of our study reveal the molecular basis of REV7 (also known as MAD2L2). Apart from REV7, the other three the SHLD3–REV7 interaction and provide critical insights into Shieldin components had not been characterized before. Among how SHLD3 recognizes REV7. these subunits, SHLD3 is the most apical element (16, 19, 22) that bridges REV7 to RIF1 and fills the gap in the 53BP1–RIF1–REV7 3 axis. Therefore, SHLD3 and REV7 form a sub-complex, which is DNA double-strand breaks (DSBs) are extremely vicious considered as the DSB-recruitment module, while SHLD2, along DNA lesions that may cause carcinogenesis or cell death if not with SHLD1, functions as the ssDNA-binding module within the complex (16). Knockdown of SHLD3 largely reduced the localiza- This work was supported by Chinese Ministry of Science and Technology Grant 2019YFA0508902 (to Z. Z.), Natural Science Foundation of China tion intensities of REV7/SHLD2/SHLD1 to the ionizing radiation- Grants 31970621 (to S. S.) and 31521002 and 31671344 (to Z. Z.), Strategic induced foci region, highlighting its RIF1-proximal role (16, 19). Priority Research Program Grant XDB0 8010104 (to Z. Z.), Ovarian Cancer Importantly, depletion of SHLD3 dampens NHEJ-related events, Research Fund Alliance Grant 605101 (to Z. G.), and National Institutes of Health NCI Grant CA222195 (to Z. G.). The authors declare that they have activates DNA end resection, and restores HR in a BRCA1-mu- no conflicts of interest with the contents of this article. The content is solely tated context, thus rendering these cells poly(ADP-ribose) poly- the responsibility of the authors and does not necessarily represent the merase inhibitors–resistant (15, 16, 19). Although SHLD3 is official views of the National Institutes of Health. essential for recruitment and function of Shieldin by directly inter- This article contains Figs. S1–S4. The atomic coordinates and structure factors (codes 6K07 and 6K08) have been acting with REV7, the underlying molecular mechanism remains deposited in the Protein Data Bank (http://wwpdb.org/). elusive. 1 To whom correspondence may be addressed. E-mail: [email protected]. 2 To whom correspondence may be addressed. E-mail: [email protected]. 3 The abbreviations used are: DSB, double-strand break; RBD, REV7-binding DNA; RBM, REV7-binding motif; IMAC, immobilized metal-affinity domain; PDB, Protein Data Bank; NHEJ, nonhomologous end-joining; HR, chromatography; Ni-NTA, nickel-nitrilotriacetic acid; IPTG, isopropyl ␤-D- homologous recombination; ITC, isothermal titration calorimetry; MBP, thiogalactoside; TLS, translesion DNA synthesis; CAMP, chromosome maltose-binding protein; SPR, surface plasmon resonance; RMSD, root alignment–maintaining phosphoprotein; SFB, S protein, FLAG, and mean square deviation; ␣C-helix, C-terminal ␣-helix; ssDNA, single-strand streptavidin-binding peptide tag. 250 J. Biol. Chem. (2020) 295(1) 250–262 © 2020 Dai et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Structural insights into REV7 recruitment by SHLD3 In this study, we report two crystal structures of REV7 in affinity as SHLD3(29–83) with a dissociation constant of complex with the SHLD3–RBD. The complex structure shows 15.7 Ϯ 0.8 nM in ITC measurement (Fig. 1b), indicating that the that SHLD3–RBD forms a “ladle” configuration when it binds amino acid residues ranging from 29 to 45 of SHLD3, although to REV7, comprising the N-terminal loop and the C-terminal conserved, are dispensable for REV7 interaction. Therefore, we ␣-helix (␣C-helix) of SHLD3–RBD, which constitute the cor- named residues 46–74 of SHLD3 as the RBD and purified the responding “stem” and “base” part of the ladle-shaped struc- REV7–SHLD3 (RBD) complex for the next round of crystal ture. The N-terminal loop is structurally similar to other REV7- screening. Encouragingly, the REV7–SHLD3 (RBD) complex binding proteins due to the presence of a well-established yielded diffraction-quality crystals. REV7-binding motif, but the ␣C-helix displays a binding mode distinct from known REV7-binding partners. Biochemical anal- Overall structure of the SHLD3–REV7 complex yses, coupled with thermodynamic measurements, have veri- The structures of the REV7R124A–SHLD3 (RBD) and fied their contribution to REV7 binding. Importantly, we dem- REV7R124A, A135D–SHLD3 (RBD) complex were determined by onstrated that the ␣C-helix region of SHLD3–RBD is a crucial the molecular replacement method using 2.2 and 2.3 Å resolu- contributing factor of robust SHLD3–REV7 interaction. Addi- tion data, respectively. Both crystals contain one single copy of tionally, we present the first kinetic view on the “safety-belt” the SHLD3–REV7 complex per asymmetric unit, and residues binding mechanism underlying the SHLD3–REV7 complex via 9–207 of REV7 and 49–73 of SHLD3 were successfully mod- surface plasmon resonance (SPR) assays. In sum, this work is eled, whereas other residues were not included in the final mod- Downloaded from the first structural study toward the role of REV7 in DSB repair els due to indiscernible electron density. The crystallographic and provides structural insights into how the DSB recruitment and geometric statistics of the refined models are exhibited in module assembles within the Shieldin complex. Table 1. Because these two structures are nearly identical, the REV7R124A–SHLD3 (RBD) structure with higher resolution Results was chosen as a representative for further description. Within the structure, REV7 displays a canonical HORMA http://www.jbc.org/ SHLD3 employs REV7-binding domain to interact with REV7 (Hop1, Rev7, and Mad2) fold and adopts the closed conforma- SHLD3 is a 250-amino acid polypeptide with a translational tion with SHLD3 peptide tightly held by the safety-belt loop of elongation initiation factor EIF4E-like domain in its C terminus REV7 (Fig. 1c). Superimposition of REV7 in our complex with (15) and contains two presumptive REV7-binding motifs the previously reported structures of REV7/REV3–RBM1 (PDB (RBM, defined by PXXXpP sequence, where X represents any code 3ABD) (24), REV7/CAMP (PDB code 5XPT) (26), and at Inst Biophys, CAS on February 23, 2020 amino acid residue and the lowercase p is a less important pro- REV7/REV3–RBM2 (PDB code 6BC8) (25) revealed overall line residue and can be replaced by an alanine in some cases) in root mean square deviation (RMSD) values of 0.830, 0.953, and the N-terminal region (Fig. 1a). Previous work has mapped the 0.915 Å, respectively, suggesting the global similarity of REV7 region (residues 29–83), which harbors the second RBM in the molecules in these protein complexes, except for some zones N-terminal region of SHLD3 that interacts with REV7 (19).

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