The mechanisms by which chromatin structure decompacts to permit access to DNA are largely unknown. Here, using a model nucleosome array system reconstituted from recombinant histone octamers we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of thè 30nm' chromatin fibre. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains stoichiometric concentrations of bound linker histone, which is essential for the formation of the `30nm' chromatin fibre. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.

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

At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.

The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1–RUVBL2–RPAP3–PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90. A 3.6 Å cryo-EM structure reveals direct interaction of a C-terminal domain of RPAP3 and the ATPase domain of RUVBL2, necessary for human R2TP assembly but absent from yeast. The mobile TPR domains of RPAP3 map to the opposite face of the ring, associating with PIH1D1, which mediates client protein recruitment. Thus, RPAP3 provides a flexible platform for bringing HSP90 into proximity with diverse client proteins.

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.

Discrete regions of the eukaryotic genome assume heritable chromatin structure that is refractory to transcription. In budding yeast, silent chromatin is characterized by the binding of the Silent Information Regulatory (Sir) proteins to unmodified nucleosomes. Using an in vitro reconstitution assay, which allows us to load Sir proteins onto arrays of regularly spaced nucleosomes, we have examined the impact of specific histone modifications on Sir protein binding and linker DNA accessibility. Two typical marks for active chromatin, H3K79 me and H4K16 ac decrease the affinity of Sir3 for chromatin, yet only H4K16 ac affects chromatin structure, as measured by nuclease accessibility. Surprisingly, we found that the Sir2-4 subcomplex, unlike Sir3, has higher affinity for chromatin carrying H4K16 ac . NAD-dependent deacetylation of H4K16ac promotes binding of the SIR holocomplex but not of the Sir2-4 heterodimer. This function of H4K16 ac cannot be substituted by H3K56 ac . We conclude that acetylated H4K16 has a dual role in silencing: it recruits Sir2-4 and repels Sir3. Moreover, the deacetylation of H4K16 ac by Sir2 actively promotes the high-affinity binding of the SIR holocomplex.

The N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown. The crystal structure of the N-terminal acetylated BAH domain of S.cerevisiae Sir3 bound to the nucleosome core particle revealed that the N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome. Additionally, we present a new method for the production of protein-nucleosome complexes for structural analysis.

The budding yeast genome contains transcriptionally repressed domains at mating-type and telomeric loci, and within rDNA repeats. Gene silencing at telomeres requires the Silent information regulators Sir2p, Sir3p, and Sir4p, whereas only the Sir2p histone deacetylase is required for rDNA repression. To understand these silencing mechanisms biochemically, we examined the subunit structure of Sir2p-containing complexes. Sir2p alone forms a stable homotrimer, whereas the SIR complex is a heterotrimer containing one copy of each Sir protein. A point mutation in the Sir2p core domain (sir2(P394L)) compromises selectively rDNA repression. This mutation impairs homotrimerization but allows SIR heterotrimer formation. Surprisingly, when sir2(P394L) is coexpressed with wild-type Sir2p, rDNA repression increases and homotrimers form. Furthermore, coexpression of sir2(P394L) and enzymatically inactive sir2(H364Y) allows crosscomplementation of rDNA repression defects. The correlation of genetic and biochemical complementation argues that Sir2p trimerization is physiologically relevant for rDNA silencing.