The initiation of DNA replication represents a committing step to cell proliferation. Appropriate replication onset depends on multiprotein complexes that help properly distinguish origin regions, generate nascent replication bubbles, and promote replisome formation. This review describes initiation systems employed by bacteria, archaea, and eukaryotes, with a focus on comparing and contrasting molecular mechanisms among organisms. Although commonalities can be found in the functional domains and strategies used to carry out and regulate initiation, many key participants have markedly different activities and appear to have evolved convergently. Despite significant advances in the field, major questions still persist in understanding how initiation programs are executed at the molecular level.
The remarkable accuracy of eukaryotic cell division is partly maintained by the cohesin complex acting as a molecular glue to prevent premature sister chromatid separation. The loading of cohesin onto chromosomes is catalyzed by the Scc2-Scc4 loader complex. Here, we report the crystal structure of Scc4 bound to the N terminus of Scc2 and show that Scc4 is a tetratricopeptide repeat (TPR) superhelix. The Scc2 N terminus adopts an extended conformation and is entrapped by the core of the Scc4 superhelix. Electron microscopy (EM) analysis reveals that the Scc2-Scc4 loader complex comprises three domains: a head, body, and hook. Deletion studies unambiguously assign the Scc2N-Scc4 as the globular head domain, whereas in vitro cohesin loading assays show that the central body and the hook domains are sufficient to catalyze cohesin loading onto circular DNA, but not chromatinized DNA in vivo, suggesting a possible role for Scc4 as a chromatin adaptor.
The basic unit of chromatin, the nucleosome core particle (NCP), controls how DNA in eukaryotic cells is compacted, replicated and read. Since its discovery, biochemists have sought to understand how this protein–DNA complex can help to control so many diverse tasks. Recent electron-microscopy (EM) studies on NCP-containing assemblies have helped to describe important chromatin transactions at a molecular level. With the implementation of recent technical advances in single-particle EM, our understanding of how nucleosomes are recognized and read looks to take a leap forward. In this review, the authors highlight recent advances in the architectural understanding of chromatin biology elucidated by EM.
SUMMARY The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. However, the mechanism of its regulation is not understood. Here, we purified human γTuRC and quantitatively characterized its nucleation properties in a TIRF microscopy-based real-time nucleation assay. We find that microtubule nucleation by γTuRC is kinetically inhibited compared to microtubule elongation. Determining the cryo-EM structure of γTuRC at 4 Å resolution reveals an asymmetric conformation with only part of the complex in a ‘closed’ conformation matching the microtubule geometry. Several factors stabilise the closed conformation. One is actin in the core of the complex and others, likely MZT1 or MZT2, line the outer perimeter of the closed part of γTuRC. The opposed side of γTuRC is in an ‘open’, nucleation-incompetent conformation, leading to a structural asymmetry, explaining the kinetic inhibition of nucleation by human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.
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.
Membrane proteins from 25 strains of B. fragilis isolated in different laboratories in Northern Italy were examined by SDS-PAGE and isoelectrofocusing. The electrophoretic patterns of inner and outer membrane after Sarkosyl and SDS solubilization of all the isolates were consistently similar to that of the reference strain. The protein profiles of the different species belonging to the B. fragilis group are clearly distinguishable with negligible similarities. Our data clearly show that this approach is extremely helpful and reliable in providing additional verification of the identity of strains recognized by conventional tests. In this connection PAGIF analysis of triton solubilized isolated envelopes reduces technical time and difficulties, thus improving analytical accuracy.
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