Backbone and ILV side-chain methyl NMR resonance assignments of human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes.

Rev7 is a versatile HORMA (Hop1, Rev7, Mad2) family adaptor protein with multiple roles in mitotic regulation and DNA damage response, and an essential accessory subunit of the translesion synthesis (TLS) DNA polymerase Polζ employed in replication of damaged DNA. Within Polζ, the two copies of Rev7 interact with the two Rev7-bonding motifs (RBM1 and RBM2) of the catalytic subunit Rev3 by a mechanism characteristic of HORMA proteins whereby the "safety-belt" loop of Rev7 closes on the top of the ligand. Here we report the nearly complete backbone and Ile, Val, Leu side-chain methyl NMR resonance assignments of the 27 kDa human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes (BMRB deposition numbers 51651 and 51652) that will facilitate future NMR studies of Rev7 dynamics and interactions.

Shieldin complex assembly kinetics and DNA binding by SHLD3.

The Shieldin complex represses end resection at DNA double-strand breaks (DSBs) and thereby serves as a pro-non homologous end joining (NHEJ) factor. The molecular details of the assembly of Shieldin and its recruitment to DSBs are unclear. Shieldin contains two REV7 molecules, which have the rare ability to slowly switch between multiple distinct native states and thereby could dynamically control the assembly of Shieldin. Here, we report the identification of a promiscuous DNA binding domain in SHLD3. At the N-terminus, SHLD3 interacts with a dimer of REV7 molecules. We show that the interaction between SHLD3 and the first REV7 is remarkably slow, while in contrast the interaction between SHLD3 and SHLD2 with a second REV7 molecule is fast and does not require structural remodeling. Overall, these results provide insights into the rate-limiting step of the molecular assembly of the Shieldin complex and its recruitment at DNA DSBs.

Evolution of Rev7 interactions in eukaryotic TLS DNA polymerase Polζ.

Translesion synthesis (TLS) DNA polymerase Polζ is crucial for the bypass replication over sites of DNA damage. The Rev7 subunit of Polζ is a HORMA (Hop1, Rev7, Mad2) protein that facilitates recruitment of Polζ to the replication fork via interactions with the catalytic subunit Rev3 and the translesion synthesis scaffold protein Rev1. Human Rev7 (hRev7) interacts with two Rev7-binding motifs (RBMs) of hRev3 by a mechanism conserved among HORMA proteins whereby the safety-belt loop of hRev7 closes on the top of the ligand. The two copies of hRev7 tethered by the two hRev3-RBMs form a symmetric head-to-head dimer through the canonical HORMA dimerization interface. Recent cryo-EM structures reveal that Saccharomyces cerevisiae Polζ (scPolζ) also includes two copies of scRev7 bound to distinct regions of scRev3. Surprisingly, the HORMA dimerization interface is not conserved in scRev7, with the two scRev7 protomers forming an asymmetric head-to-tail dimer with a much smaller interface than the hRev7 dimer. Here, we validated the two adjacent RBM motifs in scRev3, which bind scRev7 with affinities that differ by two orders of magnitude and confirmed the 2:1 stoichiometry of the scRev7:Rev3 complex in solution. However, our biophysical studies reveal that scRev7 does not form dimers in solution either on its own accord or when tethered by the two RBMs in scRev3. These findings imply that the scRev7 dimer observed in the cryo-EM structures is induced by scRev7 interactions with other Polζ subunits and that Rev7 homodimerization via the HORMA interface is a mechanism that emerged later in evolution.

Curious Case of MAD2 Protein: Diverse Folding Intermediates Leading to Alternate Native States.

Anfinsen's dogma postulates that for one sequence there will be only one unique structure that is necessary for the functioning of the protein. However, over the years there have been a number of departures from this postulate. As far as function is considered, there are growing examples of proteins that "moonlight", perform multiple unrelated functions. With the discovery of intrinsically disordered proteins, morpheeins, chameleonic sequences, and metamorphic proteins that can switch folds, we have acquired a more nuanced understanding of protein folding and dynamics. Appearing to apparently contradict the classical folding paradigm, metamorphic proteins are considered exotic species. In this work, we have explored the free energy landscape and folding pathways of the metamorphic protein MAD2 which is an important component of the spindle checkpoint. It coexists in two alternate states: the inactive open state and the active closed state. Using a dual-basin structure-based model approach we have shown that a variety of intermediates and multiple pathways are available to MAD2 to fold into its alternate forms. This approach involves performing molecular dynamics simulations of coarse-grained models of MAD2 where the structural information regarding both of its native conformations is explicitly included in terms of their native contacts in the force field used. Detailed analyses have indicated that some of the contacts within the protein play a key role in determining which folding pathway will be selected and point to a probable long-range communication between the N and the C termini of the protein that seems to control its folding. Finally, our work also provides a rationale for the experimentally observed preference of the ΔC10 variant of MAD2 to exist in the open state.

MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair.

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.

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

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.

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

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 ß 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.

Structural basis for shieldin complex subunit 3-mediated recruitment of the checkpoint protein REV7 during DNA double-strand break repair.

Shieldin complex subunit 3 (SHLD3) is the apical subunit of a recently-identified shieldin complex and plays a critical role in DNA double-strand break repair. To fulfill its function in DNA repair, SHLD3 interacts with the mitotic spindle assembly checkpoint protein REV7 homolog (REV7), but the details of this interaction remain obscure. Here, we present the crystal structures of REV7 in complex with SHLD3's REV7-binding domain (RBD) at 2.2-2.3 Å resolutions. The structures revealed that the ladle-shaped RBD in SHLD3 uses its N-terminal loop and C-terminal α-helix (αC-helix) in its interaction with REV7. The N-terminal loop exhibited a structure similar to those previously identified in other REV7-binding proteins, and the less-conserved αC-helix region adopted a distinct mode for binding REV7. In vitro and in vivo binding analyses revealed that the N-terminal loop and the αC-helix are both indispensable for high-affinity REV7 binding (with low-nanomolar affinity), underscoring the crucial role of SHLD3 αC-helix in protein binding. Moreover, binding kinetics analyses revealed that the REV7 "safety belt" region, which plays a role in binding other proteins, is essential for SHLD3-REV7 binding, as this region retards the dissociation of the RBD from the bound REV7. Together, the findings of our study reveal the molecular basis of the SHLD3-REV7 interaction and provide critical insights into how SHLD3 recognizes REV7.

REV7 has a dynamic adaptor region to accommodate small GTPase RAN/Shigella IpaB ligands, and its activity is regulated by the RanGTP/GDP switch.

REV7, also termed mitotic arrest-deficient 2-like 2 (MAD2L2 or MAD2B), acts as an interaction module in a broad array of cellular pathways, including translesion DNA synthesis, cell cycle control, and nonhomologous end joining. Numerous REV7 binding partners have been identified, including the human small GTPase Ras-associated nuclear protein (RAN), which acts as a potential upstream regulator of REV7. Notably, the Shigella invasin IpaB hijacks REV7 to disrupt cell cycle control to prevent intestinal epithelial cell renewal and facilitate bacterial colonization. However, the structural details of the REV7-RAN and REV7-IpaB interactions are mostly unknown. Here, using fusion protein and rigid maltose-binding protein tagging strategies, we determined the crystal structures of these two complexes at 2.00-2.35 Å resolutions. The structures revealed that both RAN and IpaB fragments bind the "safety belt" region of REV7, inducing rearrangement of the C-terminal ß-sheet region of REV7, conserved among REV7-related complexes. Of note, the REV7-binding motifs of RAN and IpaB each displayed some unique interactions with REV7 despite sharing consensus residues. Structural alignments revealed that REV7 has an adaptor region within the safety belt region that can rearrange secondary structures to fit a variety of different ligands. Our structural and biochemical results further indicated that REV7 preferentially binds GTP-bound RAN, implying that a GTP/GDP-bound transition of RAN may serve as the molecular switch that controls REV7's activity. These results provide insights into the regulatory mechanism of REV7 in cell cycle control, which may help with the development of small-molecule inhibitors that target REV7 activity.

[Structural Basis of the Multifunctional Hub Protein and Identification of a Small-molecule Compound for Drug Discovery].

Translesion DNA synthesis (TLS) is an emergency system activated to inhibit cell death caused by DNA damage-induced replication arrest. Thus, TLS enables cancer cells to acquire resistance to alkylate anticancer drugs. REV7 functions as the hub protein that interacts with both the inserter DNA polymerase REV1 and the extender DNA polymerase REV3 in TLS. REV7-mediated protein-protein interactions (PPIs) are essential for the activation of TLS, and are therefore attractive targets for anticancer drug development. To clarify the REV7-REV3 and REV7-REV1 PPIs, we determined the structures of REV7-REV3 and REV7-REV3-REV1 complexes. In the structures of REV7-REV3 and REV7-REV3-REV1 complexes, REV7 wraps around the REV3 fragment, and the REV1-binding interface is distinct from the REV3-binding site of REV7. We also identified a novel REV7 binding protein, transcription factor II-I (TFII-I), which is required for TLS. Of note, TFII-I binds the REV7-REV3-REV1 complex, suggesting that REV7-TFII-I PPIs are independent of other REV7-mediated PPIs. Furthermore, we found a small-molecule compound that inhibits TLS by targeting the REV7-REV3 PPIs. Lastly, we determined the structure of REV7 in complex with chromosome alignment maintaining phosphoprotein (CAMP), a known kinetochore-microtubule attachment protein. The overall structure of the REV7-CAMP complex is similar to that of the REV7-REV3 complex, but the REV7-CAMP PPIs are markedly different from the REV7-REV3 PPIs. These findings improve our understanding of multifunctional hub proteins, and are helpful for designing small-molecule compounds for novel anticancer drug development.

Rev7 dimerization is important for assembly and function of the Rev1/Polζ translesion synthesis complex.

The translesion synthesis (TLS) polymerases Polζ and Rev1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein with roles in TLS, DNA repair, and cell-cycle control, facilitates assembly of this complex by binding Rev1 and the catalytic subunit of Polζ, Rev3. Rev7 interacts with Rev3 by a mechanism conserved among HORMA proteins, whereby an open-to-closed transition locks the ligand underneath the "safety belt" loop. Dimerization of HORMA proteins promotes binding and release of this ligand, as exemplified by the Rev7 homolog, Mad2. Here, we investigate the dimerization of Rev7 when bound to the two Rev7-binding motifs (RBMs) in Rev3 by combining in vitro analyses of Rev7 structure and interactions with a functional assay in a Rev7-/- cell line. We demonstrate that Rev7 uses the conventional HORMA dimerization interface both to form a homodimer when tethered by the two RBMs in Rev3 and to heterodimerize with other HORMA domains, Mad2 and p31comet Structurally, the Rev7 dimer can bind only one copy of Rev1, revealing an unexpected Rev1/Polζ architecture. In cells, mutation of the Rev7 dimer interface increases sensitivity to DNA damage. These results provide insights into the structure of the Rev1/Polζ TLS assembly and highlight the function of Rev7 homo- and heterodimerization.

Mechanism for remodelling of the cell cycle checkpoint protein MAD2 by the ATPase TRIP13.

The maintenance of genome stability during mitosis is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex (APC/C, also known as the cyclosome)1,2. Unattached kinetochores control MCC assembly by catalysing a change in the topology of the ß-sheet of MAD2 (an MCC subunit), thereby generating the active closed MAD2 (C-MAD2) conformer3-5. Disassembly of free MCC, which is required for SAC inactivation and chromosome segregation, is an ATP-dependent process driven by the AAA+ ATPase TRIP13. In combination with p31comet, an SAC antagonist6, TRIP13 remodels C-MAD2 into inactive open MAD2 (O-MAD2)7-10. Here, we present a mechanism that explains how TRIP13-p31comet disassembles the MCC. Cryo-electron microscopy structures of the TRIP13-p31comet-C-MAD2-CDC20 complex reveal that p31comet recruits C-MAD2 to a defined site on the TRIP13 hexameric ring, positioning the N terminus of C-MAD2 (MAD2NT) to insert into the axial pore of TRIP13 and distorting the TRIP13 ring to initiate remodelling. Molecular modelling suggests that by gripping MAD2NT within its axial pore, TRIP13 couples sequential ATP-driven translocation of its hexameric ring along MAD2NT to push upwards on, and simultaneously rotate, the globular domains of the p31comet-C-MAD2 complex. This unwinds a region of the αA helix of C-MAD2 that is required to stabilize the C-MAD2 ß-sheet, thus destabilizing C-MAD2 in favour of O-MAD2 and dissociating MAD2 from p31comet. Our study provides insights into how specific substrates are recruited to AAA+ ATPases through adaptor proteins and suggests a model of how translocation through the axial pore of AAA+ ATPases is coupled to protein remodelling.

Conformational dynamics of the Hop1 HORMA domain reveal a common mechanism with the spindle checkpoint protein Mad2.

The HORMA domain is a highly conserved protein-protein interaction module found in eukaryotic signaling proteins including the spindle assembly checkpoint protein Mad2 and the meiotic HORMAD proteins. HORMA domain proteins interact with short 'closure motifs' in partner proteins by wrapping their C-terminal 'safety belt' region entirely around these motifs, forming topologically-closed complexes. Closure motif binding and release requires large-scale conformational changes in the HORMA domain, but such changes have only been observed in Mad2. Here, we show that Saccharomyces cerevisiae Hop1, a master regulator of meiotic recombination, possesses conformational dynamics similar to Mad2. We identify closure motifs in the Hop1 binding partner Red1 and in Hop1 itself, revealing that HORMA domain-closure motif interactions underlie both Hop1's initial recruitment to the chromosome axis and its self-assembly on the axis. We further show that Hop1 adopts two distinct folded states in solution, one corresponding to the previously-observed 'closed' conformation, and a second more extended state in which the safety belt region has disengaged from the HORMA domain core. These data reveal strong mechanistic similarities between meiotic HORMADs and Mad2, and provide a mechanistic basis for understanding both meiotic chromosome axis assembly and its remodeling by the AAA+ ATPase Pch2/TRIP13.

Dynamic feature of mitotic arrest deficient 2-like protein 2 (MAD2L2) and structural basis for its interaction with chromosome alignment-maintaining phosphoprotein (CAMP).

Mitotic arrest deficient 2-like protein 2 (MAD2L2), also termed MAD2B or REV7, is involved in multiple cellular functions including translesion DNA synthesis (TLS), signal transduction, transcription, and mitotic events. MAD2L2 interacts with chromosome alignment-maintaining phosphoprotein (CAMP), a kinetochore-microtubule attachment protein in mitotic cells, presumably through a novel "WK" motif in CAMP. Structures of MAD2L2 in complex with binding regions of the TLS proteins REV3 and REV1 have revealed that MAD2L2 has two faces for protein-protein interactions that are regulated by its C-terminal region; however, the mechanisms underlying the MAD2L2-CAMP interaction and the mitotic role of MAD2L2 remain unknown. Here we have determined the structures of human MAD2L2 in complex with a CAMP fragment in two crystal forms. The overall structure of the MAD2L2-CAMP complex in both crystal forms was essentially similar to that of the MAD2L2-REV3 complex. However, the residue interactions between MAD2L2 and CAMP were strikingly different from those in the MAD2L2-REV3 complex. Furthermore, structure-based interaction analyses revealed an unprecedented mechanism involving CAMP's WK motif. Surprisingly, in one of the crystal forms, the MAD2L2-CAMP complex formed a dimeric structure in which the C-terminal region of MAD2L2 was swapped and adopted an immature structure. The structure provides direct evidence for the dynamic nature of MAD2L2 structure, which in turn may have implications for the protein-protein interaction mechanism and the multiple functions of this protein. This work is the first structural study of MAD2L2 aside from its role in TLS and might pave the way to clarify MAD2L2's function in mitosis.

The kinetochore-dependent and -independent formation of the CDC20-MAD2 complex and its functions in HeLa cells.

The mitotic checkpoint complex (MCC) is formed from two sub-complexes of CDC20-MAD2 and BUBR1-BUB3, and current models suggest that it is generated exclusively by the kinetochores after nuclear envelope breakdown (NEBD). However, neither sub-complex has been visualised in vivo, and when and where they are formed during the cell cycle and their response to different SAC conditions remains elusive. Using single cell analysis in HeLa cells, we show that the CDC20-MAD2 complex is cell cycle regulated with a "Bell" shaped profile and peaks at prometaphase. Its formation begins in early prophase before NEBD when the SAC has not been activated. The complex prevents the premature degradation of cyclin B1. Tpr, a component of the NPCs (nuclear pore complexes), facilitates the formation of this prophase form of the CDC20-MAD2 complex but is inactive later in mitosis. Thus, we demonstrate that the CDC20-MAD2 complex could also be formed independently of the SAC. Moreover, in prolonged arrest caused by nocodazole treatment, the overall levels of the CDC20-MAD2 complex are gradually, but significantly, reduced and this is associated with lower levels of cyclin B1, which brings a new insight into the mechanism of mitotic "slippage" of the arrested cells.

C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast Schizosaccharomyces pombe.

The mitotic arrest deficiency 2 (Mad2) protein is an essential component of the spindle assembly checkpoint that interacts with Cdc20/Slp1 and inhibit its ability to activate anaphase promoting complex/cyclosome (APC/C). In bladder cancer cell line the C-terminal residue of the mad2 gene has been found to be deleted. In this study we tried to understand the role of the C-terminal region of mad2 on the spindle checkpoint function. To envisage the role of C-terminal region of Mad2, we truncated 25 residues of Mad2 C-terminal region in fission yeast S.pombe and characterized its effect on spindle assembly checkpoint function. The cells containing C-terminal truncation of Mad2 exhibit sensitivity towards microtubule destabilizing agent suggesting perturbation of spindle assembly checkpoint. Further, the C-terminal truncation of Mad2 exhibit reduced viability in the nda3-KM311 mutant background at non-permissive temperature. Truncation in mad2 gene also affects its foci forming ability at unattached kinetochore suggesting that the mad2-∆CT mutant is unable to maintain spindle checkpoint activation. However, in response to the defective microtubule, only brief delay of mitotic progression was observed in Mad2 C-terminal truncation mutant. In addition we have shown that the deletion of two ß strands of Mad2 protein abolishes its ability to interact with APC activator protein Slp1/Cdc20. We purpose that the truncation of two ß strands (ß7 and ß8) of Mad2 destabilize the safety belt and affect the Cdc20-Mad2 interaction leading to defects in the spindle checkpoint activation.

MAD2γ, a novel MAD2 isoform, reduces mitotic arrest and is associated with resistance in testicular germ cell tumors.

BACKGROUND: Prolonged mitotic arrest in response to anti-cancer chemotherapeutics, such as DNA-damaging agents, induces apoptosis, mitotic catastrophe, and senescence. Disruptions in mitotic checkpoints contribute resistance to DNA-damaging agents in cancer. MAD2 has been associated with checkpoint failure and chemotherapy response. In this study, a novel splice variant of MAD2, designated MAD2γ, was identified, and its association with the DNA damage response was investigated. METHODS: Endogenous expression of MAD2γ and full-length MAD2 (MAD2α) was measured using RT-PCR in cancer cell lines, normal foreskin fibroblasts, and tumor samples collected from patients with testicular germ cell tumors (TGCTs). A plasmid expressing MAD2γ was transfected into HCT116 cells, and its intracellular localization and checkpoint function were evaluated according to immunofluorescence and mitotic index. RESULTS: MAD2γ was expressed in several cancer cell lines and non-cancerous fibroblasts. Ectopically expressed MAD2γ localized to the nucleus and reduced the mitotic index, suggesting checkpoint impairment. In patients with TGCTs, the overexpression of endogenous MAD2γ, but not MAD2α, was associated with resistance to cisplatin-based chemotherapy. Likewise, cisplatin induced the overexpression of endogenous MAD2γ, but not MAD2α, in HCT116 cells. CONCLUSIONS: Overexpression of MAD2γ may play a role in checkpoint disruption and is associated with resistance to cisplatin-based chemotherapy in TGCTs.

Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling.

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.

Interaction between the Rev1 C-Terminal Domain and the PolD3 Subunit of Polζ Suggests a Mechanism of Polymerase Exchange upon Rev1/Polζ-Dependent Translesion Synthesis.

Translesion synthesis (TLS) is a mutagenic branch of cellular DNA damage tolerance that enables bypass replication over DNA lesions carried out by specialized low-fidelity DNA polymerases. The replicative bypass of most types of DNA damage is performed in a two-step process of Rev1/Polζ-dependent TLS. In the first step, a Y-family TLS enzyme, typically Polη, Polι, or Polκ, inserts a nucleotide across a DNA lesion. In the second step, a four-subunit B-family DNA polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) extends the distorted DNA primer-template. The coordinated action of error-prone TLS enzymes is regulated through their interactions with the two scaffold proteins, the sliding clamp PCNA and the TLS polymerase Rev1. Rev1 interactions with all other TLS enzymes are mediated by its C-terminal domain (Rev1-CT), which can simultaneously bind the Rev7 subunit of Polζ and Rev1-interacting regions (RIRs) from Polη, Polι, or Polκ. In this work, we identified a previously unknown RIR motif in the C-terminal part of PolD3 subunit of Polζ whose interaction with the Rev1-CT is among the tightest mediated by RIR motifs. Three-dimensional structure of the Rev1-CT/PolD3-RIR complex determined by NMR spectroscopy revealed a structural basis for the relatively high affinity of this interaction. The unexpected discovery of PolD3-RIR motif suggests a mechanism of "inserter" to "extender" DNA polymerase switch upon Rev1/Polζ-dependent TLS, in which the PolD3-RIR binding to the Rev1-CT (i) helps displace the "inserter" Polη, Polι, or Polκ from its complex with Rev1, and (ii) facilitates assembly of the four-subunit "extender" Polζ through simultaneous interaction of Rev1-CT with Rev7 and PolD3 subunits.

TRIP13 Regulates Both the Activation and Inactivation of the Spindle-Assembly Checkpoint.

Biochemical studies have indicated that p31(comet) and TRIP13 are critical for inactivating MAD2. To address unequivocally whether p31(comet) and TRIP13 are required for mitotic exit at the cellular level, their genes were ablated either individually or together in human cells. Neither p31(comet) nor TRIP13 were absolutely required for unperturbed mitosis. MAD2 inactivation was only partially impaired in p31(comet)-deficient cells. In contrast, TRIP13-deficient cells contained MAD2 exclusively in the C-MAD2 conformation. Our results indicate that although p31(comet) enhanced TRIP13-mediated MAD2 conversion, it was not absolutely necessary for the process. Paradoxically, TRIP13-deficient cells were unable to activate the spindle-assembly checkpoint, revealing that cells lacking the ability to inactivate MAD2 were incapable in mounting a checkpoint response. These results establish a paradigm of the roles of p31(comet) and TRIP13 in both checkpoint activation and inactivation.