p31^comet and TRIP13 recycle Rev7 to regulate DNA repair

Proteins of the HORMA domain family, named for its three founding members Hop1, Rev7, and Mad2, play key roles in a broad range of eukaryotic signaling pathways, from chromosome segregation and meiotic recombination, to DNA repair, to the initiation of autophagy (1). The HORMA domain nucleates assembly of multiprotein signaling complexes by wrapping its C-terminal “safety belt” region entirely around a short, 6- to 10-amino acid “closure motif” in a binding partner, resulting in a highly stable complex (Fig. 1A). While the mechanisms governing assembly of HORMA protein signaling complexes vary, many HORMA proteins share a common disassembly pathway involving two proteins, p31^comet and the AAA+ ATPase TRIP13. p31^comet, itself a diverged HORMA protein, specifically binds HORMA proteins in their “closed” partner-bound conformation, and recruits them to TRIP13 (2–4). TRIP13 partially unfolds the HORMA domain, releasing the bound closure motif and converting the substrate HORMA protein to an inactive “open” conformation poised for rebinding (5–10). This HORMA recycling pathway was first described for the spindle assembly checkpoint protein Mad2 (11–13), and has since been extended to include the meiotic recombination factor Hop1 and its relatives, collectively termed meiotic HORMADs (14–20). A key question has been whether TRIP13 and p31^comet regulate other HORMA protein families, including the DNA repair factor Rev7 and the autophagy regulators Atg13 and Atg101. In two recent manuscripts, Clairmont et al. (21) and Sarangi et al. (22) provide convincing evidence that TRIP13 and p31^comet regulate Rev7 function in two DNA repair pathways, and that overexpression of TRIP13 and/or p31^comet in cancer causes resistance to a widely used class of anticancer drugs known as Poly-ADP ribose polymerase (PARP) inhibitors.

Fig. 1.

Regulation of HORMA proteins by p31^comet and TRIP13. (A) HORMA-mediated signaling is activated when an open HORMA domain (light blue) binds a closure motif (yellow) in a binding partner and converts to the closed conformation (dark blue). To inactivate signaling, p31^comet binds a HORMA-closure motif complex and recruits it to TRIP13 for disassembly. (B) Healthy cells maintain a balance between two DNA DSB repair pathways, HR and NHEJ. When recruited to DNA ends by 53BP1/Rif1, Rev7-Shieldin inhibits DNA end resection to suppress HR. In HR-deficient BRCA1^−/− cancers, PARP inhibition leads to increased DNA breaks and cell death. Overexpression of TRIP13 and/or p31^comet in these cells reactivates HR to promote PARP inhibitor resistance, cell survival, and proliferation.

Rev7 has long been under-studied compared to Mad2 and the meiotic HORMADs, but recent years have witnessed a renaissance in our understanding of Rev7’s roles in two important DNA repair pathways: translesion DNA synthesis and DNA double-strand break (DSB) repair. Rev7 was first characterized as a subunit of DNA polymerase ζ, which aids replication of DNA past lesions that stall replicative polymerases (23, 24). A groundbreaking recent structure of the polymerase ζ holoenzyme shows that a dimer of Rev7 coordinates assembly of this complex by binding two closure motifs (also called Rev7-binding motifs or RBMs) in the catalytic Rev3 subunit, and mediating interactions with the accessory subunits Pol31 and Pol32 (25). At the same time, Rev7 binds a second DNA polymerase, Rev1, which, in turn, recruits additional Y-family DNA polymerases like Polη, Polι, and Polκ (26). In this manner, Rev7 functionally links the activity of Y-family “inserter” polymerases that insert a base directly opposite a DNA lesion with that of the “extender” polymerase Rev3 that synthesizes past the lesion for eventual handoff to a replicative polymerase.

In vertebrates, Rev7 plays a second key role in DNA repair through the newly discovered Shieldin complex, which regulates DSB repair pathway choice (27). Cells possess two major pathways for DSB repair: the homologous recombination (HR) pathway that uses an unbroken DNA template to mediate error-free repair, and the more error-prone nonhomologous end-joining (NHEJ) pathway. HR and NHEJ are tightly regulated during the cell cycle, with NHEJ dominant in G1, and HR dominant in S and G2 when a sister chromosome generated by DNA replication is available as a repair template. A key factor in repair pathway choice is the Shieldin complex, which comprises Rev7, SHLD1, SHLD2, and SHLD3. A recent structure of the Rev7−SHLD2−SHLD3 subcomplex revealed that a dimer of Rev7 nucleates Shieldin assembly through a complex set of interactions, including binding to a closure motif in SHLD3 (28). In the cell, Shieldin is recruited to DSB sites by 53BP1 and Rif1, where it inhibits DNA end resection to suppress HR and promote NHEJ (Fig. 1B) (27).

Given its central roles in two major DNA repair pathways, one important question is whether Rev7, like the related Mad2 and meiotic HORMADs, is regulated by the TRIP13/p31^comet HORMA recycling pathway. In two recent studies, Clairmont et al. (21) and Sarangi et al. (22) provide convincing evidence that TRIP13 and p31^comet regulate Rev7 through conformational recycling and Rev7-closure motif complex disassembly. The two studies show that TRIP13 and p31^comet physically interact with Rev7 and mediate disassembly of Shieldin and polymerase ζ, both in vitro and in cells. Overexpression of TRIP13 or p31^comet reduces association of Rev7 with SHLD3 and, in turn, increases DNA end resection at DSBs and promotes their repair by HR. TRIP13/p31^comet overexpression also reduces the association of Rev7 with Rev3 and impairs cells’ ability to repair DNA lesions caused by ultraviolet radiation and the interstrand crosslinker mitomycin C, demonstrating a defect in translesion DNA synthesis. These data firmly establish a role for TRIP13 and p31^comet in regulating Rev7’s function in two important DNA repair pathways.

A direct role for TRIP13 and p31^comet in Rev7 regulation can also explain these proteins’ contributions to cancer. TRIP13 is known to be overexpressed in many human cancers, and Sarangi et al. (22) now find that p31^comet overexpression is also common in cancer. Moreover, high TRIP13 and/or p31^comet levels correlate with poor prognosis across many cancer types (21, 22). While increased HORMA recycling activity in cancerous cells likely impacts many HORMA-dependent signaling pathways, Clairmont et al. (21) and Sarangi et al. (22) demonstrate that Shieldin-regulated DSB repair is of particular clinical relevance. Many cancers lose the ability to repair DNA breaks by HR through mutation or loss of BRCA1, and these cancers are sensitive to inhibition of PARP (29). Clairmont et al. (21) and Sarangi et al. (22) find that BRCA1^−/− cells overexpressing TRIP13 or p31^comet become resistant to the PARP inhibitor Olaparib, and show that this resistance arises through reactivation of HR-mediated DNA repair due to lowered Shieldin complex levels (Fig. 1B). Thus, inhibition of TRIP13/p31^comet-mediated Shieldin disassembly may represent a promising treatment strategy for patients with BRCA1-deficient cancers that develop resistance to PARP inhibitors.

Many questions remain with respect to the roles of TRIP13 and p31^comet in healthy cells and in disease. First, while Clairmont et al. (21) show that Rev7 can adopt two folded states in solution, how these two states correspond to Mad2’s well-defined open and closed conformations requires additional exploration. Another question is whether and how Rev7 recycling is regulated through the cell cycle. Two studies have reported that p31^comet is phosphorylated during mitosis to suppress disassembly of mitotic checkpoint complexes (30, 31); whether this or other regulatory mechanisms applies to Rev7 regulation is unknown. Finally, whether TRIP13 and p31^comet regulate autophagy through Atg13 and Atg101, or other signaling pathways through as yet undiscovered HORMA proteins, remains mostly unexplored. While many questions remain, the studies by Clairmont et al. (21) and Sarangi et al. (22) provide significant insight into HORMA recycling by TRIP13 and p31^comet, and clearly define a key connection between these proteins and human disease.