Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular “mix and match” approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.

The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring‐shaped protein complexes that are thought to function by entrapping DNA. It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three‐dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single‐molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA‐bound proteins and nucleosomes but is constrained in its movement by transcription and DNA‐bound CCCTC‐binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.

SUMMARY The Mitotic Checkpoint Complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated Anaphase-promoting complex/Cyclosome (APC/CCDC20). APC/CCDC20 and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/CCDC20–UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here, electron microscopy reveals conformational variation of APC/CCDC20-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly, and conformational variability of APC/C, modulate UBE2C-catalyzed ubiquitylation of MCC’s CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/CCDC20–MCC modulating E2 activation underlies distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation.