Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and ...cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.
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•3.9-Å cryo-EM structure of the cohesin complex in a DNA gripping state intermediate•DNA is trapped between two gates that lead into the cohesin ring•The kleisin N-tail guides DNA through a kleisin N-gate into the gripping state•ATP hydrolysis opens the head gate to complete DNA entry
Cohesin is a ring-shaped protein complex that topologically entraps DNA to fulfil key functions in chromosome architecture. In a multidisciplinary approach, Higashi et al. use cryo-EM and biochemical techniques to describe how ATP-fueled structural changes of the cohesin complex drive the DNA entry reaction into the cohesin ring.
In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA1,2. The mechanism of MCM ...double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading ofthe first hexamer drives the recruitment of the second hexamer3. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites4,5. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains ofthe MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data.
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 angstrom 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.
Programmed cell death is a critical process that is necessary to cull damaged cells as well as eliminate unnecessary cells during normal development. Improper regulation of this process can result in ...neurodegenerative diseases and cancer. While developmental cell death involves the death of healthy cells, most programmed cell death eliminates unhealthy cells that are unable to recover from stress. There are various different types of cellular stress that can result in cell death including oxidative and endoplasmic reticulum stress. The transparent nematode worm Caenorhabditis elegans is an ideal model in which to study programmed cell death due to its short life cycle and genetic tractability. The genes controlling cell death were originally discovered in this organism and the pathway is conserved from C. elegans to humans. Parkinson's disease is a neurodegenerative disorder that results in progressive loss of motor function. Though the majority of cases are sporadic, a small proportion are due to genetic mutations. One of the proteins involved in familial Parkinson's disease, PINK1, is a mitochondrial serine-threonine kinase that protects mitochondria from oxidative stress and aids in the initiation of mitophagy. Loss of PINK1 function results in increased cell death in humans and Drosophila. In C. elegans PINK-1 instead functions as an activator of programmed cell death. This pro-apoptotic function is due to the N-terminal domain rather than kinase function. The accumulation of misfolded proteins in the endoplasmic reticulum results in programmed cell death if the unfolded protein response fails to resolve the stress. In C. elegans loss of ICD-1, the homolog of βNAC, results in excessive ectopic cell death. Loss of ICD-1 function results in the inappropriate targeting of nascent peptides to the endoplasmic reticulum resulting in misfolded protein stress. The cell death induced is completely dependent on CED-4, the Apaf1 homolog, but is more dependent on both the canonical caspase CED-3 in the early-stage embryos and the non-canonical caspase CSP-1 in late-stage embryos. Under stress calcium can be released by the endoplasmic reticulum into the cytoplasm. The calpain CLP-2, a calcium activated protease, is also necessary for late-stage embryonic cell deaths in ICD-1 depleted animals.
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