Nse5/6 is a negative regulator of the ATPase activity of the Smc5/6 complex.

The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein 'holo-complex' is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase activity, binding to the head-end of the complex to suppress turnover, irrespective of the DNA-bound status of the complex.

Purified Smc5/6 Complex Exhibits DNA Substrate Recognition and Compaction.

Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring.

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.

Cryo-EM structures of holo condensin reveal a subunit flip-flop mechanism.

Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding yeast Saccharomyces cerevisiae. Our structures of the five-subunit condensin holo complex at different functional stages suggest that ATP binding induces the transition of the SMC coiled coils from a folded-rod conformation into a more open architecture. ATP binding simultaneously triggers the exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA-binding sites in the catalytic core of condensin, forming the basis of the DNA translocation and loop-extrusion activities.

Cryo-EM structure of SWI/SNF complex bound to a nucleosome.

The chromatin-remodelling complex SWI/SNF is highly conserved and has critical roles in various cellular processes, including transcription and DNA-damage repair1,2. It hydrolyses ATP to remodel chromatin structure by sliding and evicting histone octamers3-8, creating DNA regions that become accessible to other essential factors. However, our mechanistic understanding of the remodelling activity is hindered by the lack of a high-resolution structure of complexes from this family. Here we report the cryo-electron microscopy structure of Saccharomyces cerevisiae SWI/SNF bound to a nucleosome, at near-atomic resolution. In the structure, the actin-related protein (Arp) module is sandwiched between the ATPase and the rest of the complex, with the Snf2 helicase-SANT associated (HSA) domain connecting all modules. The body contains an assembly scaffold composed of conserved subunits Snf12 (also known as SMARCD or BAF60), Snf5 (also known as SMARCB1, BAF47 or INI1) and an asymmetric dimer of Swi3 (also known as SMARCC, BAF155 or BAF170). Another conserved subunit, Swi1 (also known as ARID1 or BAF250), resides in the core of SWI/SNF, acting as a molecular hub. We also observed interactions between Snf5 and the histones at the acidic patch, which could serve as an anchor during active DNA translocation. Our structure enables us to map and rationalize a subset of cancer-related mutations in the human SWI/SNF complex and to propose a model for how SWI/SNF recognizes and remodels the +1 nucleosome to generate nucleosome-depleted regions during gene activation9.

Organization of the Escherichia coli Chromosome by a MukBEF Axial Core.

Structural maintenance of chromosomes (SMC) complexes organize chromosomes ubiquitously, thereby contributing to their faithful segregation. We demonstrate that under conditions of increased chromosome occupancy of the Escherichia coli SMC complex, MukBEF, the chromosome is organized as a series of loops around a thin (<130 nm) MukBEF axial core, whose length is ∼1,100 times shorter than the chromosomal DNA. The linear order of chromosomal loci is maintained in the axial cores, whose formation requires MukBEF ATP hydrolysis. Axial core structure in non-replicating chromosomes is predominantly linear (1 µm) but becomes circular (1.5 µm) in the absence of MatP because of its failure to displace MukBEF from the 800 kbp replication termination region (ter). Displacement of MukBEF from ter by MatP in wild-type cells directs MukBEF colocalization with the replication origin. We conclude that MukBEF individualizes and compacts the chromosome lengthwise, demonstrating a chromosome organization mechanism similar to condensin in mitotic chromosome formation.

Promotion of Hyperthermic-Induced rDNA Hypercondensation in Saccharomyces cerevisiae.

Ribosome biogenesis is tightly regulated through stress-sensing pathways that impact genome stability, aging and senescence. In Saccharomyces cerevisiae, ribosomal RNAs are transcribed from rDNA located on the right arm of chromosome XII. Numerous studies reveal that rDNA decondenses into a puff-like structure during interphase, and condenses into a tight loop-like structure during mitosis. Intriguingly, a novel and additional mechanism of increased mitotic rDNA compaction (termed hypercondensation) was recently discovered that occurs in response to temperature stress (hyperthermic-induced) and is rapidly reversible. Here, we report that neither changes in condensin binding or release of DNA during mitosis, nor mutation of factors that regulate cohesin binding and release, appear to play a critical role in hyperthermic-induced rDNA hypercondensation. A candidate genetic approach revealed that deletion of either HSP82 or HSC82 (Hsp90 encoding heat shock paralogs) result in significantly reduced hyperthermic-induced rDNA hypercondensation. Intriguingly, Hsp inhibitors do not impact rDNA hypercondensation. In combination, these findings suggest that Hsp90 either stabilizes client proteins, which are sensitive to very transient thermic challenges, or directly promotes rDNA hypercondensation during preanaphase. Our findings further reveal that the high mobility group protein Hmo1 is a negative regulator of mitotic rDNA condensation, distinct from its role in promoting premature condensation of rDNA during interphase upon nutrient starvation.

Cryo-EM structures of remodeler-nucleosome intermediates suggest allosteric control through the nucleosome.

The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeFx that capture two potential reaction intermediates. In one structure, histone residues near the dyad and in the H2A-H2B acidic patch, distal to the active SNF2h protomer, appear disordered. The disordered acidic patch is expected to inhibit the second SNF2h protomer, while disorder near the dyad is expected to promote DNA translocation. The other structure doesn't show octamer deformation, but surprisingly shows a 2 bp translocation. FRET studies indicate that ADP-BeFx predisposes SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric octamer deformation to inhibit the second protomer, while stimulating directional DNA translocation.

Condensins and cohesins - one of these things is not like the other!

Condensins and cohesins are highly conserved complexes that tether together DNA loci within a single DNA molecule to produce DNA loops. Condensin and cohesin structures, however, are different, and the DNA loops produced by each underlie distinct cell processes. Condensin rods compact chromosomes during mitosis, with condensin I and II complexes producing spatially defined and nested looping in metazoan cells. Structurally adaptive cohesin rings produce loops, which organize the genome during interphase. Cohesin-mediated loops, termed topologically associating domains or TADs, antagonize the formation of epigenetically defined but untethered DNA volumes, termed compartments. While condensin complexes formed through cis-interactions must maintain chromatin compaction throughout mitosis, cohesins remain highly dynamic during interphase to allow for transcription-mediated responses to external cues and the execution of developmental programs. Here, I review differences in condensin and cohesin structures, and highlight recent advances regarding the intramolecular or cis-based tetherings through which condensins compact DNA during mitosis and cohesins organize the genome during interphase.

The involvement of tau in nucleolar transcription and the stress response.

Tau is known for its pathological role in neurodegenerative diseases, including Alzheimer's disease (AD) and other tauopathies. Tau is found in many subcellular compartments such as the cytosol and the nucleus. Although its normal role in microtubule binding is well established, its nuclear role is still unclear. Here, we reveal that tau localises to the nucleolus in undifferentiated and differentiated neuroblastoma cells (SHSY5Y), where it associates with TIP5, a key player in heterochromatin stability and ribosomal DNA (rDNA) transcriptional repression. Immunogold labelling on human brain sample confirms the physiological relevance of this finding by showing tau within the nucleolus colocalises with TIP5. Depletion of tau results in an increase in rDNA transcription with an associated decrease in heterochromatin and DNA methylation, suggesting that under normal conditions tau is involved in silencing of the rDNA. Cellular stress induced by glutamate causes nucleolar stress associated with the redistribution of nucleolar non-phosphorylated tau, in a similar manner to fibrillarin, and nuclear upsurge of phosphorylated tau (Thr231) which doesn't colocalise with fibrillarin or nucleolar tau. This suggests that stress may impact on different nuclear tau species. In addition to involvement in rDNA transcription, nucleolar non-phosphorylated tau also undergoes stress-induced redistribution similar to many nucleolar proteins.

Combining in Vitro and in Silico Single-Molecule Force Spectroscopy to Characterize and Tune Cellulosomal Scaffoldin Mechanics.

Cellulosomes are polyprotein machineries that efficiently degrade cellulosic material. Crucial to their function are scaffolds consisting of highly homologous cohesin domains, which serve a dual role by coordinating a multiplicity of enzymes as well as anchoring the microbe to its substrate. Here we combined two approaches to elucidate the mechanical properties of the main scaffold ScaA of Acetivibrio cellulolyticus. A newly developed parallelized one-pot in vitro transcription-translation and protein pull-down protocol enabled high-throughput atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) measurements of all cohesins from ScaA with a single cantilever, thus promising improved relative force comparability. Albeit very similar in sequence, the hanging cohesins showed considerably lower unfolding forces than the bridging cohesins, which are subjected to force when the microbe is anchored to its substrate. Additionally, all-atom steered molecular dynamics (SMD) simulations on homology models offered insight into the process of cohesin unfolding under force. Based on the differences among the individual force propagation pathways and their associated correlation communities, we designed mutants to tune the mechanical stability of the weakest hanging cohesin. The proposed mutants were tested in a second high-throughput AFM SMFS experiment revealing that in one case a single alanine to glycine point mutation suffices to more than double the mechanical stability. In summary, we have successfully characterized the force induced unfolding behavior of all cohesins from the scaffoldin ScaA, as well as revealed how small changes in sequence can have large effects on force resilience in cohesin domains. Our strategy provides an efficient way to test and improve the mechanical integrity of protein domains in general.

Crystal structure of human PCNA in complex with the PIP box of DVC1.

In higher eukaryotes, DVC1 (SPRTN, Spartan or C1orf124) is implicated in the translesion synthesis (TLS) pathway. DVC1 localizes to sites of DNA damage, binds to the proliferating cell nuclear antigen (PCNA) via its conserved PCNA-interacting motif (PIP box), and associates with ubiquitin selective segregase p97 and other factors, thus regulating translesion synthesis polymerases. Here, we report the crystal structure of human PCNA in complex with a peptide ((321)SNSHQNVLSNYFPRVS(336)) derived from human DVC1 that contains a unique YF type PIP box. Structural analysis reveals the detailed PIP box-PCNA interaction. Interestingly, substitution of Y331 with Phe severely reduces its PCNA binding affinity. These findings offer new insights into the determinants of PIP box for PCNA binding.

Chromosome Scaffold is a Double-Stranded Assembly of Scaffold Proteins.

Chromosome higher order structure has been an enigma for over a century. The most important structural finding has been the presence of a chromosome scaffold composed of non-histone proteins; so-called scaffold proteins. However, the organization and function of the scaffold are still controversial. Here, we use three dimensional-structured illumination microscopy (3D-SIM) and focused ion beam/scanning electron microscopy (FIB/SEM) to reveal the axial distributions of scaffold proteins in metaphase chromosomes comprising two strands. We also find that scaffold protein can adaptably recover its original localization after chromosome reversion in the presence of cations. This reversion to the original morphology underscores the role of the scaffold for intrinsic structural integrity of chromosomes. We therefore propose a new structural model of the chromosome scaffold that includes twisted double strands, consistent with the physical properties of chromosomal bending flexibility and rigidity. Our model provides new insights into chromosome higher order structure.

Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion.

Orderly termination of sister-chromatid cohesion during mitosis is critical for accurate chromosome segregation. During prophase, mitotic kinases phosphorylate cohesin and its protector sororin, triggering Wapl-dependent cohesin release from chromosome arms. The shugoshin (Sgo1)-PP2A complex protects centromeric cohesin until its cleavage by separase at anaphase onset. Here, we report the crystal structure of a human cohesin subcomplex comprising SA2 and Scc1. Multiple HEAT repeats of SA2 form a dragon-shaped structure. Scc1 makes extensive contacts with SA2, with one binding hotspot. Sgo1 and Wapl compete for binding to a conserved site on SA2-Scc1. At this site, mutations of SA2 residues that disrupt Wapl binding bypass the Sgo1 requirement in cohesion protection. Thus, in addition to recruiting PP2A to dephosphorylate cohesin and sororin, Sgo1 physically shields cohesin from Wapl. This unexpected, direct antagonism between Sgo1 and Wapl augments centromeric cohesion protection.

Nuclear structures surrounding internal lamin invaginations.

A- and C-type lamins are intermediate filament proteins responsible for the maintenance of nuclear shape and most likely nuclear architecture. Here, we propose that pronounced invaginations of A/C-type lamins into the nuclear interior represent channels for the transport of regulatory molecules to and from nuclear and nucleolar regions. Using fluorescent protein technology and immunofluorescence, we show that A-type lamin channels interact with several nuclear components, including fibrillarin- and UBF-positive regions of nucleoli, foci of heterochromatin protein 1 ß, polycomb group bodies, and genomic regions associated with DNA repair. Similar associations were observed between A/C-type lamin channels and nuclear pores, lamin-associated protein LAP2α, and promyelocytic leukemia nuclear bodies. Interestingly, regions with high levels of A/C-type lamins had low levels of B-type lamins, and vice versa. These characteristics were observed in primary and immortalized mouse embryonic fibroblasts as well as human and mouse embryonic stem cell colonies exhibiting stem cell-specific lamin positivity. Our findings indicate that internal channels formed by nuclear lamins likely contribute to normal cellular processes through association with various nuclear and nucleolar structures.

A two-step mechanism for epigenetic specification of centromere identity and function.

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

[Non-histone skeleton of mitotic chromosome in situ].

Studying giant nuclei of Chironomus plumosus in situ (Makarov, Chentsov, 2010), we concluded that polythene chromosome structure appears after 2 M NaCl and DNase treatment in presence of 2 mM CuCl2. Cu2+ -ions may stabilize bonds between specific non-histone components, arranged into non-histone matrix of polythene chromosome. Here, we investigated the non-histone matrix of pig embryo mitotic chromosomes in situ, using 2 mM CuCl2-stabilization method. In 2 mM CuCl2-stabilized cells the residual chromosome body (non-histone matrix) could be visualized in every stage of mitosis. Mitotic chromosome non-histone matrix had the same reaction on preliminary hypotonic treatment as normal chromosome: different decondensation of non-histone material was observed. Topoisomerase IIalpha and SMC 1 had uniform localization inside chromosomal body and did not form any axial structures.

[Nuclear protein matrix from giant nuclei of Chironomus plumosus determinates polythene chromosome organization].

Giant nuclei from salivary glands of Chironomus plumosus were treated in situ with detergent, 2 M NaCl and nucleases in order to reveal residual nuclear matrix proteins (NMP). It was shown, that preceding stabilization of non-histone proteins with 2 mM CuCl2 allowed to visualize the structure of polythene chromosomes at every stage of the extraction of histones and DNA. Stabilized NPM of polythene chromosomes maintains their morphology and banding patterns, which is observed by light and electron microscopy, whereas internal fibril net or residual nucleoli are not found. In stabilized NPM of polythene chromosomes, topoisomerase IIalpha and SMC1 retain their localization that is typical of untreated chromosomes. NPM of polythene chromosomes also includes sites of DNA replication, visualized with BrDU incubation, and some RNA-components. So, we can conclude that structure of NPM from giant nuclei is equal to NPM from normal interphase nuclei, and that morphological features of polythene chromosomes depend on the presence of NMP.

Pre-Lamin A processing is linked to heterochromatin organization.

Pre-lamin A undergoes subsequent steps of post-translational modification at its C-terminus, including farnesylation, methylation, and cleavage by ZMPSTE24 metalloprotease. Here, we show that accumulation of different intermediates of pre-lamin A processing in nuclei, induced by expression of mutated pre-lamin A, differentially affected chromatin organization in human fibroblasts. Unprocessed (non-farnesylated) pre-lamin A accumulated in intranuclear foci, caused the redistribution of LAP2alpha and of the heterochromatin markers HP1alpha and trimethyl-K9-histone 3, and triggered heterochromatin localization in the nuclear interior. In contrast, the farnesylated and carboxymethylated lamin A precursor accumulated at the nuclear periphery and caused loss of heterochromatin markers and Lap2alpha in enlarged nuclei. Interestingly, pre-lamin A bound both HP1alpha and LAP2alpha in vivo, but the farnesylated form showed reduced affinity for HP1alpha. Our data show a link between pre-lamin A processing and heterochromatin remodeling and have major implications for understanding molecular mechanisms of human diseases linked to mutations in lamins.

Structural insight into the interaction between the p55 PDZ domain and glycophorin C.

p55, a member of the membrane-associated guanylate kinase family, includes a PDZ domain that specifically interacts with the C-terminal region of glycophorin C in the ternary complex of p55, protein 4.1 and glycophorin C. Here we present the first NMR-derived complex structure of the p55 PDZ domain and the C-terminal peptide of glycophorin C, obtained by using a threonine to cysteine (T85C) mutant of the p55 PDZ domain and a phenylalanine to cysteine (F127C) mutant of the glycophorin C peptide. Our NMR results revealed that the two designed mutant molecules retain the specific interaction manner that exists between the wild type molecules and can facilitate the structure determination by NMR, due to the stable complex formation via an intermolecular disulfide bond. The complex structure provides insight into the specific interaction of the p55 PDZ domain with the two key residues, Ile128 and Tyr126, of glycophorin C.