Silencing the mitotic checkpoint requires that new MCC formation is prevented and pre-existing MCC is disassembled. This will release the APC/C-Cdc20 from inhibition so it can resume its activity and promote anaphase onset. Very recent studies

have hypothesized that the joint action of the AAA+ ATPase TRIP13 and an adapter protein p31comet can mediate the catalytic disassociation of the mitotic checkpoint protein Mad2 from Cdc20-Mad2 complexes or from the four protein complex BubR1-Bub3-Mad2-Cdc20 known as the mitotic checkpoint complex (MCC). Furthermore, using purified components in vitro, TRIP13 ATPase-dependent activity has been shown to convert closed, “active” Mad2, capable of binding to Cdc20, into its inactive “open” conformer. This is only in conjunction with p31comet, which is proposed to act as an adapter to facilitate recognition of closed Mad2 and deliver it to TRIP13. Viewing all evidence, key mechanistic questions are unresolved, most specifically the role(s) of TRIP13 and p31comet in mitotic checkpoint silencing. An ATP-hydrolysis-deficient mutant of TRIP13 (E253Q), is shown to stably capture its substrates, as its ATP-hydrolysis activity is required for release and remodeling of its substrates (48). By utilizing this mutant we can view TRIP13 substrate binding and elucidate TRIP13 function. Using all purified components, I performed disassembly assays to identify whether TRIP13 and p31comet can disassemble both the initiator of MCC, the Mad1-Mad2 template, and a pre-existing potent inhibitor of the APC/C-Cdc20, the MCC. We can conclude that the joint activity of TRIP13 and p31comet, in the presence of ATP, do not disassemble Mad2 from the Mad1-Mad2 core complex. Additionally, the MCC disassembly assay shows good potential to allow us to discern if there is a role for TRIP13 and p31comet in disassembling the MCC