Structure of the cohesin loader Scc2

Chao, William Chong Hang; Murayama, Yasuto; Muñoz, Sofía; Jones, Andrew W.; Wade, B.O.; Purkiss, A.; Hu, Xiaowen; Borg, Annabel; Snijders, Ambrosius P.; Uhlmann, Frank; Singleton, Martin R.

doi:10.1038/ncomms13952

Cited by 49 publications

(63 citation statements)

References 52 publications

(108 reference statements)

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“…A 3D reconstruction revealed two extended protrusions, in the shape of a bean and a rod respectively, which asymmetrically depart from the core of the complex . A U-shaped density was apparent as part of the core, reminiscent of the A. gossypii and C. thermophilum cohesin loader subunit Scc2 (Chao et al, 2017;Kikuchi et al, 2016). Other cohesin components and the DNA were harder to assign.…”

Section: Cryo-em Structure Of Cohesin In the Gripping Statementioning

confidence: 99%

“…Regions where the Mis4 cryo-EM density is connected with DNA include R487 and R874, where the N-terminal handle and the C-terminal hook clamp the double helix ( Figure 3B). Two conserved positively charged residues, K873 and K877, map in close proximity and are required for cohesin loading onto chromosomes in S. cerevisiae (Chao et al, 2017). The engaged SMC heads provide an additional, composite DNA binding surface, lined with conserved positively charged residues on both Psm1 and Psm3 ( Figure S3A).…”

Section: The Molecular Action Of the Cohesin Loadermentioning

confidence: 99%

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

Higashi

Eickhoff²,

Simões³

et al. 2020

Preprint

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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 biophysical approaches and 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 biochemical experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an Nterminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against a shut ATPase gate. ATP hydrolysis leads to ATPase gate opening to complete DNA entry. Whether DNA loading is successful, or rather 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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Section: Cryo-em Structure Of Cohesin In the Gripping Statementioning

confidence: 99%

Section: The Molecular Action Of the Cohesin Loadermentioning

confidence: 99%

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

Higashi

Eickhoff²,

Simões³

et al. 2020

Preprint

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“…8D). It is interesting to note that the identified NIPBL domains containing RNA binding activities are not conserved in lower eukaryotes in contrast to the conserved cohesin and Scc4 interacting domains (Chao et al, 2015;Chao et al, 2017;Hinshaw et al, 2015;Kikuchi et al, 2016) (Fig. 5C).…”

Section: Nipbl In Stress Responsementioning

confidence: 94%

The cohesin loader NIPBL interacts with pre-ribosomal RNA and treacle to regulate ribosomal RNA synthesis

Kong

Chen

Lin

et al. 2019

Preprint

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NIPBL is an essential loader of cohesin to mediate sister chromatid cohesion and chromatin loop organization. NIPBL mutations cause Cornelia de Lange Syndrome. How NIPBL's genomic localization is specified is not fully understood. We found that NIPBL localizes to the nucleolus in an RNA-dependent manner and binds directly to ribosomal RNA (rRNA). We identified two RNA binding domains in NIPBL in vitro, both of which are required for efficient rRNA binding in vivo. NIPBL binds to ribosomal DNA (rDNA) in an RNA-stimulated manner, recruits PAF1 and promotes pre-rRNA transcription. Stress that inhibits rRNA synthesis displaces NIPBL from the nucleolus and rDNA. Interestingly, treacle, mutated in Treacher Collins syndrome, tightly binds to and recruits NIPBL to the nucleolus, nucleolar organizer regions, and the stress-induced nucleolar cap. The results reveal that a subpopulation of NIPBL is recruited to the nucleolus through its interaction with RNA and treacle and regulates pre-rRNA transcription.6 Results NIPBL localizes to the Fibrillar Center of the nucleolusThe nucleolus has a "tripartite structure" consisting of the Fibrillar Center (FC), the Dense Fibrillar Component (DFC), and the Granular Component (GC), each with distinct functions (Fig. 1A) (Boulon et al., 2010a). The FC is the site for pre-rRNA transcription and is enriched in RNA polymerase I (Pol I) machinery. Pre-rRNA is processed in the DFC, which contains many RNA processing factors such as snoRNA and fibrillarin. The GC is the site where pre-ribosome assembly takes place, and contains ribosome assembly factors such as B23 (nucleophosmin). Consistent with cohesin binding to the rDNA region (Zeng et al., 2009a), biochemical fractionation analysis indicates that Nipbl and cohesin are present in both nuclear and nucleolar fractions (Fig. 1B). Using an affinity-purified polyclonal antibody directed against the C-terminus of human NIPBL, we observed clear localization of both mouse Nipbl and human NIPBL in the nucleolus (mouse embryonic fibroblasts (MEFs) in Fig. 1C and D; human 293T cells in Supplemental Fig. S1, respectively). Costaining with antibodies specific for B23 (a marker for the GC) and fibrillarin (a marker for the DFC) revealed that Nipbl tightly clusters to the inside of the DFC corresponding to the FC (Fig. 1C and D). Furthermore, nucleolar Nipbl staining signals were reduced significantly in a Nipbl heterozygous mutant MEFs (modeling NIPBL haploinsufficiency in CdLS (Kawauchi et al., 2009;Newkirk et al., 2017)) and Nipbl siRNAdepleted MEFs compared to the wild type and control siRNA-treated MEFs, respectively (Fig. 1E). These results confirmed the specificity of immunostaining and also indicate that Nipbl in the nucleolus is sensitive to partial protein depletion (haploinsufficiency). NIPBL localizes to the nucleolus in an RNA-dependent manner and binds to preribosomal RNARNA is highly enriched and is an important constituent of nucleoli. Interestingly, RNase treatment dispersed Nipbl nucleolar foci while bulk Nipbl remained in the nucl...

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“…MultiBac was first adopted by the structural biology community and has enabled structure elucidation of many multiprotein complexes, illuminating their cellular functions. Here, we show a selection of recent structures obtained with MultiBac-produced specimens [22][23][24][25][26][27][28][29][30][31] (Fig. 2).…”

Section: Accelerating Multiprotein Complex Structural Biologymentioning

confidence: 99%

“…Multiprotein complex structural biology. A selection of recent near-atomic structures of MultiBac-produced specimens, determined by X-ray crystallography or electron cryo-microscopy[22][23][24][25][26][27][28][29][30][31]. In the case of mTOR complex 1, heterologous Raptor was produced by using MultiBac and crystallized, and the crystal coordinates fitted into a cryo-EM map of endogenously purified holo-complex in a hybrid approach[31].…”

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confidence: 99%

MultiBac: from protein complex structures to synthetic viral nanosystems

et al. 2017

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The MultiBac baculovirus/insect cell expression vector system was conceived as a user-friendly, modular tool-kit for producing multiprotein complexes for structural biology applications. MultiBac has allowed the structure and function of many molecular machines to be elucidated, including previously inaccessible high-value drug targets. More recently, MultiBac developments have shifted to customized baculoviral genomes that are tailored for a range of applications, including synthesizing artificial proteins by genetic code expansion. We review some of these developments, including the ongoing rewiring of the MultiBac system for mammalian applications, notably CRISPR/Cas9-mediated gene editing.

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Structure of the cohesin loader Scc2

Cited by 49 publications

References 52 publications

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

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

The cohesin loader NIPBL interacts with pre-ribosomal RNA and treacle to regulate ribosomal RNA synthesis

MultiBac: from protein complex structures to synthetic viral nanosystems

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