Publications
151 results found
Ayala R, Willhoft O, Aramayo R, et al., 2018, Structure and regulation of the human INO80–nucleosome complex, Nature, Vol: 556, Pages: 391-395, ISSN: 0028-0836
Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines1. Biochemical studies2-4 have placed the motor domains of several remodellers on the superhelical location (SHL) 2 region of the nucleosome. Structural studies on Chd1 and Snf2 (RSC) in complex with nucleosomes5-7 have provided insights into the basic mechanism of nucleosome sliding by these complexes. However, how larger, multi-subunit remodelling complexes, such as INO80, interact with nucleosomes or how remodellers carry out functions such as nucleosome sliding8, histone exchange9, and nucleosome spacing10-12 remains poorly understood. Although some remodellers work as monomers13, others work as highly cooperative dimers11,14,15. Here we present the structure of the INO80 chromatin remodeller with a bound nucleosome revealing that INO80 interacts with nucleosomes in a unique manner with the motor domains located at the entry point to the wrap around the histone core rather than at SHL2. The Arp5-Ies6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This unique arrangement allows the H3 tails of the nucleosome to play a role in regulation, differing from other characterised remodellers.
Zhang X, Aramayo RJ, Willhoft O, et al., 2017, CryoEM structures of the human INO80 chromatin remodelling complex, Nature Structural and Molecular Biology, Vol: 25, Pages: 37-44, ISSN: 1545-9985
Access to chromatin for processes such as DNA repair and transcription requires the sliding of nucleosomes along DNA. The multi-subunit INO80 chromatin remodelling complex has a particular role in DNA repair. Here we present the cryo electron microscopy structures of the active core complex of human INO80 at 9.6 Å with portions at 4.1 Å resolution along with reconstructions of combinations of subunits. Together these structures reveal the architecture of the INO80 complex, including Ino80 and actin-related proteins, which is assembled around a single Tip49a (RUVBL1) and Tip49b (RUVBL2) AAA+ heterohexamer. An unusual spoked-wheel structural domain of the Ino80 subunit is engulfed by this heterohexamer and the intimate association of this Ino80 domain with the heterohexamer is at the core of the complex. We also identify a cleft in RUVBL1 and RUVBL2, which forms a major interaction site for partner proteins and likely communicates partner-interactions with its nucleotide binding sites.
Glyde R, Ye F, Darbari V, et al., 2017, Structures of RNA polymerase closed and intermediate complexes revealmechanisms of DNA opening and transcription initiation, Molecular Cell, Vol: 67, Pages: 106-116, ISSN: 1097-2765
Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo.
Wigley DB, Willhoft O, McCormack EA, et al., 2017, Cross-talk within a functional INO80 complex dimer regulates nucleosome sliding, eLife, Vol: 6, ISSN: 2050-084X
Several chromatin remodellers have the ability to space nucleosomes on DNA. For ISWI remodellers, this involves an interplay between H4 histone tails, the AutoN and NegC motifs of the motor domains that together regulate ATPase activity and sense the length of DNA flanking the nucleosome. By contrast, the INO80 complex also spaces nucleosomes but is not regulated by H4 tails and lacks the AutoN and NegC motifs. Instead nucleosome sliding requires cooperativity between two INO80 complexes that monitor DNA length simultaneously on either side of the nucleosome during sliding. The C-terminal domain of the human Ino80 subunit (Ino80CTD) binds cooperatively to DNA and dimerisation of these domains provides crosstalk between complexes. ATPase activity, rather than being regulated, instead gradually becomes uncoupled as nucleosome sliding reaches an end point and this is controlled by the Ino80CTD. A single active ATPase motor within the dimer is sufficient for sliding.
Sawicka M, Aramayo R, Ayala R, et al., 2017, Single-Particle Electron Microscopy Analysis of DNA Repair Complexes, Methods in Enzymology, Vol: 592, Pages: 159-186, ISSN: 0076-6879
DNA repair complexes play crucial roles in maintaining genome integrity, which is essential for the survival of an organism. The understanding of their modes of action is often obscure due to limited structural knowledge. Structural characterizations of these complexes are often challenging due to a poor protein production yield, a conformational flexibility, and a relatively high molecular mass. Single-particle electron microscopy (EM) has been successfully applied to study some of these complexes as it requires low amount of samples, is not limited by the high molecular mass of a protein or a complex, and can separate heterogeneous assemblies. Recently, near-atomic resolution structures have been obtained with EM owing to the advances in technology and image processing algorithms. In this chapter, we review the EM methodology of obtaining three-dimensional reconstructions of macromolecular complexes and provide a workflow that can be applied to DNA repair complex assemblies.
Zhang N, Darbari VC, Glyde R, et al., 2016, The bacterial enhancer-dependent RNA polymerase, Biochemical Journal, Vol: 473, Pages: 3741-3753, ISSN: 1470-8728
Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript.
von Nicolai C, Ehlén Å, Martin C, et al., 2016, A second DNA binding site in human BRCA2 promotes homologous recombination., Nature Communications, Vol: 7, Pages: 12813-12813, ISSN: 2041-1723
BRCA2 tumour-suppressor protein is well known for its role in DNA repair by homologous recombination (HR); assisting the loading of RAD51 recombinase at DNA double-strand breaks. This function is executed by the C-terminal DNA binding domain (CTD) which binds single-stranded (ss)DNA, and the BRC repeats, which bind RAD51 and modulate its assembly onto ssDNA. Paradoxically, analysis of cells resistant to DNA damaging agents missing the CTD restore HR proficiency, suggesting another domain may take over its function. Here, we identify a region in the N terminus of BRCA2 that exhibits DNA binding activity (NTD) and provide evidence for NTD promoting RAD51-mediated HR. A missense variant detected in breast cancer patients located in the NTD impairs HR stimulation on dsDNA/ssDNA junction containing substrates. These findings shed light on the function of the N terminus of BRCA2 and have implications for the evaluation of breast cancer variants.
Guo J, Liang J, Zhang X, et al., 2016, Reliability Analysis of MMCs Considering Submodule Designs with Individual or Series-Operated IGBTs, IEEE TRANSACTIONS ON POWER DELIVERY, Vol: 32, Pages: 666-677, ISSN: 0885-8977
The half-bridge-based modular multilevel converter (MMC) has emerged as the favored converter topology for voltage-source HVDC applications. The submodules within the converter can be constructed with either individual insulated-gate bipolar transistor (IGBT) modules or with series-connected IGBTs, which allows for different redundancy strategies to be employed. The main contribution of this paper is that an analytical method was proposed to analyze the reliability of MMCs with the consideration of submodule arrangements and redundancy strategies. Based on the analytical method, the relative merits of two approaches to adding redundancy, and variants created by varying the submodule voltage, are assessed in terms of overall converter reliability. Case studies were conducted to compare the reliability characteristics of converters constructed using the two submodule topologies. It is found that reliability of the MMC with series-connected IGBTs is higher for the first few years but then decreases rapidly. By assigning a reduced nominal voltage to the series valve submodule upon IGBT module failure, the need to install redundant submodules is greatly reduced.
Zhang N, Jovanovic G, McDonald C, et al., 2016, Transcription regulation and membrane stress management in enterobacterial pathogens, Advances in Experimental Medicine and Biology, Vol: 915, Pages: 207-230, ISSN: 0065-2598
Transcription regulation in a temporal and conditional manner underpins the lifecycle of enterobacterial pathogens. Upon exposure to a wide array of environmental cues, these pathogens modulate their gene expression via the RNA polymerase and associated sigma factors. Different sigma factors, either involved in general 'house-keeping' or specific responses, guide the RNA polymerase to their cognate promoter DNAs. The major alternative sigma54 factor when activated helps pathogens manage stresses and proliferate in their ecological niches. In this chapter, we review the function and regulation of the sigma54-dependent Phage shock protein (Psp) system-a major stress response when Gram-negative pathogens encounter damages to their inner membranes. We discuss the recent development on mechanisms of gene regulation, signal transduction and stress mitigation in light of different biophysical and biochemical approaches.
Sawicka M, Wanrooij PH, Darbari VC, et al., 2016, The dimeric architecture of checkpoint kinases Mec1ATR and Tel1ATM reveal a common structural organisation., Journal of Biological Chemistry, Vol: 291, Pages: 13436-13447, ISSN: 1083-351X
The phosphatidylinositol 3-kinase-related protein kinases (PIKKs) are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1-Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signalling and repair. Here, we present the first structural insight for dimers of Mec1-Ddc2 and Tel1 using single particle electron microscopy. Both kinases reveal a head-to-head dimer with one major dimeric interface through their N-terminal HEAT repeats. Their dimeric interface is significantly distinct from the interface of mTOR Complex 1 dimer, which oligomerises through two spatially separate interfaces. We also observe different structural organisation of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1-Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated providing potential access of substrates to this kinase, even in its dimeric form.
Zhang N, Schaefer J, Sharma A, et al., 2015, Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis, Journal of Molecular Biology, Vol: 427, Pages: 3516-3526, ISSN: 1089-8638
In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions are differentially required during σ70-dependent and the contrasting σ54-dependent transcription activations and thus potentially underlie the key mechanistic differences between the two transcription paradigms. We further demonstrate that the transcription factor DksA directly regulates σ54-dependent activation both positively and negatively. This finding is consistent with the observed impacts of DksA on σ70-dependent promoters. DksA does not seem to significantly affect RNAP binding to a pre-melted promoter DNA but affects extensively activity at the stage of initial RNA synthesis on σ54-regulated promoters. Strikingly, removal of the σ54 Region I is sufficient to invert the action of DksA (from stimulation to inhibition or vice versa) at two test promoters. The RNAP mutants we generated also show a strong propensity to backtrack. These mutants increase the rate of transcript-hydrolysis cleavage to a level comparable to that seen in the Thermus aquaticus RNAP even in the absence of a non-complementary nucleotide. These novel phenotypes imply an important function of the bridge helix and switch regions as an anti-backtracking ratchet and an RNA hydrolysis regulator.
Yang Y, Darbari VC, Zhang N, et al., 2015, Structures of the RNA polymerase-sigma(54) reveal new and conserved regulatory strategies, Science, Vol: 349, Pages: 882-885, ISSN: 0036-8075
Transcription by RNA polymerase (RNAP) in bacteria requires specific promoter recognition by σ factors. The major variant σ factor (σ54) initially forms a transcriptionally silent complex requiring specialized adenosine triphosphate–dependent activators for initiation. Our crystal structure of the 450-kilodalton RNAP-σ54 holoenzyme at 3.8 angstroms reveals molecular details of σ54 and its interactions with RNAP. The structure explains how σ54 targets different regions in RNAP to exert its inhibitory function. Although σ54 and the major σ factor, σ70, have similar functional domains and contact similar regions of RNAP, unanticipated differences are observed in their domain arrangement and interactions with RNAP, explaining their distinct properties. Furthermore, we observe evolutionarily conserved regulatory hotspots in RNAPs that can be targeted by a diverse range of mechanisms to fine tune transcription.
Bush M, Ghosh T, Sawicka M, et al., 2015, The structural basis for enhancer-dependent assembly and activation of the AAA transcriptional activator NorR, MOLECULAR MICROBIOLOGY, Vol: 95, Pages: 17-30, ISSN: 0950-382X
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- Citations: 12
Shahid T, Soroka J, Kong EH, et al., 2014, Structure and mechanism of action of the BRCA2 breast cancer tumor suppressor, Nature Structural and Molecular Biology, Vol: 21, Pages: 962-968, ISSN: 1545-9985
Mutations in BRCA2 increase susceptibility to breast, ovarian and prostate cancers. The product of human BRCA2, BRCA2 protein, has a key role in the repair of DNA double-strand breaks and interstrand cross-links by RAD51-mediated homologous recombination. Here, we present a biochemical and structural characterization of full-length (3,418 amino acid) BRCA2, alone and in complex with RAD51. We show that BRCA2 facilitates nucleation of RAD51 filaments at multiple sites on single-stranded DNA. Three-dimensional EM reconstructions revealed that BRCA2 exists as a dimer and that two oppositely oriented sets of RAD51 molecules bind the dimer. Single-stranded DNA binds along the long axis of BRCA2, such that only one set of RAD51 monomers can form a productive complex with DNA and establish filament formation. Our data define the molecular mechanism by which this tumor suppressor facilitates RAD51-mediated homologous-recombinational repair.
Liu J, Yang J, Wen J, et al., 2014, Mutational analysis of dimeric linkers in peri- and cytoplasmic domains of histidine kinase DctB reveals their functional roles in signal transduction, OPEN BIOLOGY, Vol: 4, ISSN: 2046-2441
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- Citations: 10
Ewens CA, Panico S, Kloppsteck P, et al., 2014, The p97-FAF1 Protein Complex Reveals a Common Mode of p97 Adaptor Binding, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 289, Pages: 12077-12084
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- Citations: 20
Sharma A, Leach RN, Gell C, et al., 2014, Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies, NUCLEIC ACIDS RESEARCH, Vol: 42, Pages: 5177-5190, ISSN: 0305-1048
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- Citations: 18
Yeung HO, Foerster A, Bebeacua C, et al., 2014, Inter-ring rotations of AAA ATPase p97 revealed by electron cryomicroscopy, Open Biology, Vol: 4, ISSN: 2046-2441
The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes.
Darbari VC, Lawton E, Lu D, et al., 2014, Molecular basis of nucleotide-dependent substrate engagement and remodeling by an AAA plus activator, NUCLEIC ACIDS RESEARCH, Vol: 42, Pages: 9249-9261, ISSN: 0305-1048
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- Citations: 1
Saravanan M, Wuerges J, Bose D, et al., 2012, Interactions between the nucleosome histone core and Arp8 in the INO80 chromatin remodeling complex, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 109, Pages: 20883-20888, ISSN: 0027-8424
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- Citations: 40
Fillet S, Daniels C, Pini C, et al., 2012, Transcriptional control of the main aromatic hydrocarbon efflux pump in <i>Pseudomonas</i>, ENVIRONMENTAL MICROBIOLOGY REPORTS, Vol: 4, Pages: 158-167, ISSN: 1758-2229
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- Citations: 17
Niwa H, Ewens CA, Tsang C, et al., 2012, The Role of the N-Domain in the ATPase Activity of the Mammalian AAA ATPase p97/VCP, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 287, Pages: 8561-8570
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- Citations: 90
Bebeacua C, Förster A, McKeown C, et al., 2012, Distinct conformations of the protein complex p97-Ufd1-Npl4 revealed by electron cryomicroscopy., Proc Natl Acad Sci U S A, Vol: 109, Pages: 1098-1103
p97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to p97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with p97. Our reconstructions show that p97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the p97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by p97 and also explains how p97-Ufd1-Npl4 could form other complexes in a hierarchical model of p97-cofactor assembly.
Joly N, Zhang N, Buck M, et al., 2012, Coupling AAA protein function to regulated gene expression, BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, Vol: 1823, Pages: 108-116, ISSN: 0167-4889
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- Citations: 28
Kloppsteck P, Ewens CA, Foerster A, et al., 2012, Regulation of p97 in the ubiquitin-proteasome system by the UBX protein-family, BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, Vol: 1823, Pages: 125-129, ISSN: 0167-4889
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- Citations: 55
Fillet S, Krell T, Morel B, et al., 2011, Intramolecular signal transmission in a tetrameric repressor of the IclR family, Proceedings of the National Academy of Sciences of USA, Vol: 37, Pages: 15372-15377
Bush M, Ghosh T, Tucker N, et al., 2011, Transcriptional regulation by the dedicated nitric oxide sensor, NorR: a route towards NO detoxification, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 39, Pages: 289-293, ISSN: 0300-5127
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- Citations: 31
Klein BJ, Bose D, Baker KJ, et al., 2011, RNA polymerase and transcription elongation factor Spt4/5 complex structure, Proc Natl Acad Sci U S A., Vol: 108(2), Pages: 546-550
Jovanovic M, Burrows PC, Bose D, et al., 2011, Activity map of the Escherichia coli RNA polymerase bridge helix, J Biol Chem, Vol: 286, Pages: 14469-14479, ISSN: 1083-351X
Transcription, the synthesis of RNA from a DNA template, is performed by multisubunit RNA polymerases (RNAPs) in all cellular organisms. The bridge helix (BH) is a distinct feature of all multisubunit RNAPs and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and RNA and DNA binding. Because the BH has been captured in both kinked and straight conformations in different crystals structures of RNAP, recently supported by molecular dynamics studies, it has been proposed that cycling between these conformations is an integral part of the nucleotide addition cycle. To further evaluate the role of the BH, we conducted systematic alanine scanning mutagenesis of the Escherichia coli RNAP BH to determine its contributions to activities required for transcription. Combining our data with an atomic model of E. coli RNAP, we suggest that alterations in the interactions between the BH and (i) the trigger loop, (ii) fork loop 2, and (iii) switch 2 can help explain the observed changes in RNAP functionality associated with some of the BH variants. Additionally, we show that extensive defects in E. coli RNAP functionality depend upon a single previously not studied lysine residue (Lys-781) that is strictly conserved in all bacteria. It appears that direct interactions made by the BH with other conserved features of RNAP are lost in some of the E. coli alanine substitution variants, which we infer results in conformational changes in RNAP that modify RNAP functionality.
Alguel Y, Lu D, Quade N, et al., 2010, Crystal structure of MexZ, a key repressor responsible for antibiotic resistance in <i>Pseudomonas aeruginosa</i>, JOURNAL OF STRUCTURAL BIOLOGY, Vol: 172, Pages: 305-310, ISSN: 1047-8477
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- Citations: 27
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