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Disassembly of the Shieldin Complex by TRIP13.

ABSTRACT: In the past decade, the study of the major DNA double strand break (DSB) repair pathways, homologous recombination (HR) and classical non-homologous end joining (C-NHEJ), has revealed a vast and intricate network of regulation. The choice between HR and C-NHEJ is largely controlled at the step of DNA end-resection. A pro-C-NHEJ cascade commencing with 53BP1 and culminating in the newly discovered REV7-Shieldin complex impedes end resection and therefore HR. Importantly, loss of any component of this pathway confers PARP inhibitor resistance in BRCA1-deficient cells; hence, their study is of great clinical importance. The newest entrant on the scene of end resection regulation is the ATPase TRIP13 that disables the pro-C-NHEJ cascade by promoting a novel conformational change of the HORMA protein REV7. Here, we tie these new findings and factors with previous research on the regulation of DSB repair and HORMA proteins, and suggest testable hypotheses for how TRIP13 could specifically inactivate REV7-Shieldin to promote HR. We also discuss these biological questions in the context of clinical therapeutics.

SUBMITTER: Sarangi P

PROVIDER: S-EPMC7480811 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Molecular mechanisms of assembly and TRIP13-mediated remodeling of the human Shieldin complex.

Project description: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.

| S-EPMC7923543 | biostudies-literature

MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair.

Project description:MAD2L2 (REV7) plays an important role in DNA double-strand break repair. As a member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization-surface, HORMA-domain protein MAD2L2 has the extraordinary ability to wrap its C-terminus around SHLD3, likely creating a very stable complex. We show that appropriate function of MAD2L2 within shieldin requires its dimerization, mediated by SHLD2 and accelerating MAD2L2-SHLD3 interaction. Dimerization-defective MAD2L2 impairs shieldin assembly and fails to promote NHEJ. Moreover, MAD2L2 dimerization, along with the presence of SHLD3, allows shieldin to interact with the TRIP13 ATPase, known to drive topological switches in HORMA-domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity.

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The shieldin complex mediates 53BP1-dependent DNA repair.

Project description:53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

| S-EPMC6141009 | biostudies-literature

MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair

Project description:MAD2L2 (also known as REV7) functions in a diverse range of processes. It facilitates DNA lesion bypass through translesion synthesis (TLS), aids in interstrand crosslink repair and contributes to timely mitotic progression. Moreover, MAD2L2 plays an important role in DNA repair at DNA double strand breaks (DSBs) and uncapped telomeres. As the first identified member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice between non-homologous end-joining (NHEJ) and homologous recombination (HR) by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization surface, HORMA domain containing protein MAD2L2 has the extraordinary ability to wrap its C-terminal tail around an interacting peptide motif in SHLD3, thereby likely creating a very stable complex. We show that appropriate function of MAD2L2 in DNA repair as a member of the shieldin complex requires it to form a dimer that is mediated by SHLD2 and accelerates MAD2L2-SHLD3 interaction. Dimerization defective MAD2L2 impairs assembly of shieldin and fails to promote NHEJ at telomeres and during immunoglobulin class switch recombination (CSR). Moreover, dimerization of MAD2L2, along with the presence of SHLD3, allows the shieldin complex to interact with the AAA+ family ATPase TRIP13, known to drive topological switches in HORMA domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity.

2021-09-21 | PXD026912 | Pride

Shieldin complex whole-genome CRISPR screen

Project description:Shieldin complex promotes DNA end joining and counters homologous recombination in BRCA1-null cells

| PRJEB26796 | ENA

Disassembly of mitotic checkpoint complexes by the joint action of the AAA-ATPase TRIP13 and p31(comet).

Project description:The mitotic (or spindle assembly) checkpoint system delays anaphase until all chromosomes are correctly attached to the mitotic spindle. When the checkpoint is active, a Mitotic Checkpoint Complex (MCC) assembles and inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C). MCC is composed of the checkpoint proteins Mad2, BubR1, and Bub3 associated with the APC/C activator Cdc20. When the checkpoint signal is turned off, MCC is disassembled and the checkpoint is inactivated. The mechanisms of the disassembly of MCC are not sufficiently understood. We have previously observed that ATP hydrolysis is required for the action of the Mad2-binding protein p31(comet) to disassemble MCC. We now show that HeLa cell extracts contain a factor that promotes ATP- and p31(comet)-dependent disassembly of a Cdc20-Mad2 subcomplex and identify it as Thyroid Receptor Interacting Protein 13 (TRIP13), an AAA-ATPase known to interact with p31(comet). The joint action of TRIP13 and p31(comet) also promotes the release of Mad2 from MCC, participates in the complete disassembly of MCC and abrogates checkpoint inhibition of APC/C. We propose that TRIP13 plays centrally important roles in the sequence of events leading to MCC disassembly and checkpoint inactivation.

| S-EPMC4142996 | biostudies-literature

53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ.

Project description:53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination1,2, and reduced fidelity of long-range V(D)J recombination3. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres4, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis5-7. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive8, unlike 53BP1-deficient cells9,10, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin-a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)-as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.

| S-EPMC6989217 | biostudies-literature

Molecular basis for assembly of the shieldin complex and its implications for NHEJ.

Project description:Shieldin, including SHLD1, SHLD2, SHLD3 and REV7, functions as a bridge linking 53BP1-RIF1 and single-strand DNA to suppress the DNA termini nucleolytic resection during non-homologous end joining (NHEJ). However, the mechanism of shieldin assembly remains unclear. Here we present the crystal structure of the SHLD3-REV7-SHLD2 ternary complex and reveal an unexpected C (closed)-REV7-O (open)-REV7 conformational dimer mediated by SHLD3. We show that SHLD2 interacts with O-REV7 and the N-terminus of SHLD3 by forming β sheet sandwich. Disruption of the REV7 conformational dimer abolishes the assembly of shieldin and impairs NHEJ efficiency. The conserved FXPWFP motif of SHLD3 binds to C-REV7 and blocks its binding to REV1, which excludes shieldin from the REV1/Pol ζ translesion synthesis (TLS) complex. Our study reveals the molecular architecture of shieldin assembly, elucidates the structural basis of the REV7 conformational dimer, and provides mechanistic insight into orchestration between TLS and NHEJ.

| S-EPMC7181697 | biostudies-literature

Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells.

Project description:BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.

| S-EPMC6145444 | biostudies-literature

Membrane lipids influence protein complex assembly-disassembly.

Project description:Approximately 11% smaller t-/v-SNARE ring complexes are generated using 50 nm cholesterol-associated vesicles as opposed to vesicles containing L-alpha-lysophosphatidylcholine (LPC), as observed using atomic force microscopy. Circular dichroism spectroscopy demonstrated that in the presence of LPC as opposed to cholesterol, N-ethylmaleimide-sensitive factor + adenosine triphosphate induces disassembly of beta-sheet structures but not the alpha-helical contents within the t-/v-SNARE complex.

| S-EPMC2862647 | biostudies-literature

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