Highlights d The g-tubulin ring complex (gTuRC) nucleates microtubules and caps their minus ends d Microtubule nucleation from purified gTuRC is highly cooperative, yet inefficient d A partly open, asymmetric structure of gTuRC explains inefficient nucleation d Actin and MZT2 stabilize the closed part of the gTuRC structure

Highlights d 3.9-Å cryo-EM structure of the cohesin complex in a DNA gripping state intermediate d DNA is trapped between two gates that lead into the cohesin ring d The kleisin N-tail guides DNA through a kleisin N-gate into the gripping state d ATP hydrolysis opens the head gate to complete DNA entry

In preparation for bidirectional replication, the origin recognition complex (ORC) loads two MCM helicases forming a head-to-head double hexamer (DH) around DNA 1,2. How DH formation occurs is debated. Single-molecule experiments suggest a sequential mechanism whereby ORCdependent loading of the first hexamer drives second hexamer recruitment 3. In contrast, biochemical data show that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites 4,5. We visualized MCM loading using time-resolved EM, to identify DH formation intermediates. We confirm that both hexamers are recruited via the same interaction between the MCM and ORC C-terminal domains, and identify the mechanism for coupled MCM loading. A first loaded hexamer locked around DNA is recognized by ORC, which unexpectedly engages the N-terminal homo-dimerization interface of MCM. In this configuration, ORC is poised to direct second hexamer recruitment in an inverted orientation, suitable for DH formation. Our data reconcile two apparently contrasting models. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 Å resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors.

SummaryIn the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools single-stranded DNA through its central channel. Not all six ATPase sites are required for unwinding; however, the helicase mechanism is unknown. We imaged ATP-hydrolysis-driven translocation of the CMG using cryo-electron microscopy (cryo-EM) and found that the six MCM subunits engage DNA using four neighboring protomers at a time, with ATP binding promoting DNA engagement. Morphing between different helicase states leads us to suggest a non-symmetric hand-over-hand rotary mechanism, explaining the asymmetric requirements of ATPase function around the MCM ring of the CMG. By imaging of a higher-order replisome assembly, we find that the Mrc1-Csm3-Tof1 fork-stabilization complex strengthens the interaction between parental duplex DNA and the CMG at the fork, which might support the coupling between DNA translocation and fork unwinding.