Abstract
The Shieldin complex, composed of REV7, SHLD1, SHLD2, and SHLD3, protects DNA double-strand breaks (DSBs) to promote nonhomologous end joining. The AAA+ ATPase TRIP13 remodels Shieldin to regulate DNA repair pathway choice. Here we report crystal structures of human SHLD3-REV7 binary and fused SHLD2-SHLD3-REV7 ternary complexes, revealing that assembly of Shieldin requires fused SHLD2-SHLD3 induced conformational heterodimerization of open (O-REV7) and closed (C-REV7) forms of REV7. We also report the cryogenic electron microscopy (cryo-EM) structures of the ATPγS-bound fused SHLD2-SHLD3-REV7-TRIP13 complexes, uncovering the principles underlying the TRIP13-mediated disassembly mechanism of the Shieldin complex. We demonstrate that the N terminus of REV7 inserts into the central channel of TRIP13, setting the stage for pulling the unfolded N-terminal peptide of C-REV7 through the central TRIP13 hexameric channel. The primary interface involves contacts between the safety-belt segment of C-REV7 and a conserved and negatively charged loop of TRIP13. This process is mediated by ATP hydrolysis-triggered rotatory motions of the TRIP13 ATPase, thereby resulting in the disassembly of the Shieldin complex.
Highlights
| | Shieldin assembly TRIP13-mediated disassembly of Shieldin SHLD3–REV7 | | complex SHLD2–SHLD3–REV7 complex SHLD2–SHLD3–REV7–TRIP13 complex
We report on X-ray and cryogenic electron microscopy (cryo-EM) structural studies on the assembly of the human Shieldin complex composed of SHLD2, SHLD3, and REV7, as well as its complex with bound TRIP13 toward understanding the principles underlying TRIP13-mediated disassembly of Shieldin
We report on several structures of fused SHLD2–SHLD3 bound to REV7 in the absence and presence of TRIP13
Summary
| | Shieldin assembly TRIP13-mediated disassembly of Shieldin SHLD3–REV7 | | complex SHLD2–SHLD3–REV7 complex SHLD2–SHLD3–REV7–TRIP13 complex. A crystal structure of the SHLD3 fragment–REV7(R124A) binary complex confirms that monomeric REV7 capitalizes on its stereotypical “safety belt,” a common structural feature of HORMA family proteins, to encircle SHLD3 [26] Another crystal structure of Rev bound to fragments of SHLD2 and SHLD3 has provided insights into the assembly of this Shieldin trimeric subcomplex [27]. TRIP13 forms a hexamer to drive the REV7-mediated disassembly of Shieldin in an adenosine triphosphate (ATP)-dependent manner, thereby releasing the DSB DNA ends for resection to promote HDR [29,30,31]. The AAA+ ATPase TRIP13 captures the N terminus of C-REV7 (REVNT) within its central hexameric channel, with the rotatory motion associated with sequential ATP hydrolysis within individual TRIP13 subunits This facilitates the stepwise pulling of the REV7NT through the central channel, resulting in initial disassembly of C-REV7 followed by dissociation of the Shieldin complex
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