Imperial College London

Professor Dale Wigley FRS

Faculty of MedicineDepartment of Medicine

Chair in Protein Crystallography
 
 
 
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Contact

 

+44 (0)20 7594 8417d.wigley

 
 
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Assistant

 

Miss Kelly Butler +44 (0)20 7594 2763

 
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Location

 

258Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

16 results found

Wilkinson M, Drabavicius G, Silanskas A, Gasiunas G, Siksnys V, Wigley Det al., 2019, Structure of the DNA-bound spacer capture complex of a Type II CRISPR-Cas system., Molecular Cell, ISSN: 1097-2765

CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2–6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.

JOURNAL ARTICLE

Wilkinson OJ, Martín-González A, Kang H, Northall SJ, Wigley DB, Moreno-Herrero F, Dillingham MSet al., 2019, CtIP forms a tetrameric dumbbell-shaped particle which bridges complex DNA end structures for double-strand break repair, eLife, Vol: 8, ISSN: 2050-084X

CtIP is involved in the resection of broken DNA during the S and G2 phases of the cell cycle for repair by recombination. Acting with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with complex structures including forked ssDNA overhangs and nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.

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Cheng K, Wigley DB, 2018, DNA translocation mechanism of an XPD family helicase, eLife, Vol: 7, ISSN: 2050-084X

The XPD family of helicases, that includes human disease-related FANCJ, DDX11 and RTEL1, are Superfamily 2 helicases that contain an iron-sulphur cluster domain, translocate on ssDNA in a 5'-3' direction and play important roles in genome stability. Consequently, mutations in several of these family members in eukaryotes cause human diseases. Family members in bacteria, such as the DinG helicase from Escherichia coli, are also involved in DNA repair. Here we present crystal structures of complexes of DinG bound to single-stranded DNA (ssDNA) in the presence and absence of an ATP analogue (ADP•BeF3), that suggest a mechanism for 5'-3' translocation along the ssDNA substrate. This proposed mechanism has implications for how those enzymes of the XPD family that recognise bulky DNA lesionsmight stall at these as the first step in initiating DNA repair. Biochemical data reveal roles for conserved residues that are mutated in human diseases.

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Willhoft O, Ghoneim M, Lin C-L, Chua E, Wilkinson M, Chaban Y, Ayala R, McCormack E, Ocloo L, Rueda D, Wigley DBet al., 2018, Structure and dynamics of the yeast SWR1:nucleosome complex, Science, Vol: 362, ISSN: 0036-8075

INTRODUCTIONCanonical nucleosomes contain two copies of each of four histone proteins: H2A, H2B, H3, and H4. However, variants of these histones can be inserted by adenosine triphosphate (ATP)–dependent chromatin-remodeling machines. The yeast SWR1 chromatin-remodeling complex, a member of the INO80 remodeler family, catalyzes the exchange of H2A-H2B dimers for dimers containing Htz1 (H2A.Z in human) in an ATP-dependent manner. However, the mechanism by which SWR1 exchanges histones is poorly understood. Despite having a DNA translocase subunit similar to that in the INO80 complex that slides nucleosomes, no net translocation of nucleosomes has been reported for SWR1. Consequently, the function of the ATPase activity, which is required for histone exchange in SWR1, has remained enigmatic.RATIONALETo obtain sufficient quantities for structural analysis, we generated the complete 14-subunit yeast SWR1 complex in insect cells. Binding of nucleosomes to SWR1 is stabilized in the presence of an ATP analog (ADP•BeF3), which we used to prepare a complex with a canonical yeast H2A-containing nucleosome. Structural analysis was undertaken by cryo–electron microscopy (cryo-EM). We also used single-molecule FRET (smFRET) techniques to probe the dynamics of nucleosomes bound to SWR1. Fluorescent probes were positioned on the H2A histones and the end of the DNA to monitor changes in nucleosome dynamics upon binding of SWR1 and ATP (or ATP analogs).RESULTSWe determined the cryo-EM structure of the SWR1-nucleosome complex at 3.6-Å resolution. The architecture of the complex shows how the SWR1 complex is assembled around a heterohexameric core of the RuvBL1 and RuvBL2 subunits. The Swr1 motor subunit binds at superhelical location 2 (SHL2), a position it shares in common with other remodelers but not with its most closely related complex, INO80, which binds at SHL6-SHL7. Binding of ATP or ADP•BeF3 to the SWR1-nucleosome complex induces substantial unwrap

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Ayala R, Willhoft O, Aramayo R, Wilkinson M, McCormack E, Ocloo L, Wigley D, Zhang Xet 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.

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Zhang X, Aramayo RJ, Willhoft O, Ayala R, Bythell-Douglas R, Wigley DBet 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.

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Wigley DB, Willhoft O, McCormack EA, Aramayo R, Bythell-Douglas R, Ocloo L, Zhang Xet 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.

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Lin C-L, Chaban Y, Rees DM, McCormack EA, Ocloo L, Wigley DBet al., 2017, Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex, Nucleic Acids Research, Vol: 45, Pages: 7249-7260, ISSN: 1362-4962

We have prepared recombinant fourteen subunit yeast SWR1 complex from insect cells using a modified MultiBac system. The 1.07 MDa recombinant protein complex has histone-exchange activity. Full exchange activity is realized with a single SWR1 complex bound to a nucleosome. We also prepared mutant complexes that lack a variety of subunits or combinations of subunits and these start to reveal roles for some of these subunits as well as indicating interactions between them in the full complex. Complexes containing a series of N-terminally and C-terminally truncated Swr1 subunits reveal further details about interactions between subunits as well as their binding sites on the Swr1 subunit. Finally, we present electron microscopy studies revealing the dynamic nature of the complex and a 21 Å resolution reconstruction of the intact complex provides details not apparent in previously reported structures, including a large central cavity of sufficient size to accommodate a nucleosome.

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Sanders K, Lin C-L, Smith AJ, Cronin N, Fisher G, Eftychidis V, McGlynn P, Savery NJ, Wigley DB, Dillingham MSet al., 2017, The structure and function of an RNA polymerase interaction domain in the PcrA/UvrD helicase, Nucleic Acids Research, Vol: 45, Pages: 3875-3887, ISSN: 0305-1048

The PcrA/UvrD helicase functions in multiple pathways that promote bacterial genome stability including the suppression of conflicts between replication and transcription and facilitating the repair of transcribed DNA. The reported ability of PcrA/UvrD to bind and backtrack RNA polymerase (1,2) might be relevant to these functions, but the structural basis for this activity is poorly understood. In this work, we define a minimal RNA polymerase interaction domain in PcrA, and report its crystal structure at 1.5 Å resolution. The domain adopts a Tudor-like fold that is similar to other RNA polymerase interaction domains, including that of the prototype transcription-repair coupling factor Mfd. Removal or mutation of the interaction domain reduces the ability of PcrA/UvrD to interact with and to remodel RNA polymerase complexes in vitro. The implications of this work for our understanding of the role of PcrA/UvrD at the interface of DNA replication, transcription and repair are discussed.

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Rees DM, Willhoft O, Lin C-L, Bythell-Douglas R, Wigley DBet al., 2017, Production and Assay of Recombinant Multisubunit Chromatin Remodeling Complexes, DNA REPAIR ENZYMES: STRUCTURE, BIOPHYSICS, AND MECHANISM, Vol: 592, Pages: 27-47, ISSN: 0076-6879

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Wilkinson M, Troman L, Wan Nur Ismah WA, Chaban Y, Avison M, Dillingham M, Wigley DBet al., 2016, Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones, eLife, Vol: 5, ISSN: 2050-084X

Our previous paper (Wilkinson et al, 2016) used high-resolution cryo-electron microscopy to solve the structure of the Escherichia coli RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate. We also show that expression of Gam in E. coli or Klebsiella pneumoniae increases sensitivity to fluoroquinolones; antibacterials that kill cells by inhibiting topoisomerases and inducing double-stranded DNA breaks. Furthermore, fluoroquinolone-resistance in K. pneumoniae clinical isolates is reversed by expression of Gam. Together, our data explain the synthetic lethality observed between topoisomerase-induced DNA breaks and the RecBCD gene products, suggesting a new co-antibacterial strategy.

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Wilkinson M, Chaban Y, Wigley DB, 2016, Mechanism for nuclease regulation in RecBCD., eLife, Vol: 5, ISSN: 2050-084X

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5’-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system.

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Willhoft O, Bythell-Douglas R, Mccormack EA, Wigley DBet al., 2016, Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex, Nucleic Acids Research, Vol: 44, Pages: 8179-8188, ISSN: 1362-4962

We have purified a minimal core human Ino80 complex from recombinant protein expressedin insect cells. The complex comprises one subunit each of an N-terminally truncated Ino80,actin, Arp4, Arp5, Arp8, Ies2 and Ies6, together with a single heterohexamer of the Tip49aand Tip49b proteins. This core complex has nucleosome sliding activity that is similar to thatof endogenous human and yeast Ino80 complexes and is also inhibited by inositolhexaphosphate (IP6). We show that IP6 is a non-competitive inhibitor that acts by blockingthe stimulatory effect of nucleosomes on the ATPase activity. The IP6 binding site is locatedwithin the C-terminal region of the Ino80 subunit. We have also prepared complexes lackingcombinations of Ies2 and Arp5/Ies6 subunits that reveal regulation imposed by each of themindividually and synergistically that couples ATP hydrolysis to nucleosome sliding. Thiscoupling between Ies2 and Arp5/Ies6 can be overcome in a bypass mutation of the Arp5subunit that is active in the absence of Ies2. These studies reveal several underlyingmechanisms for regulation of ATPase activity involving a complex interplay between theseprotein subunits and IP6 that in turn controls nucleosome sliding.

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Krajewski WW, Fu X, Wilkinson M, Cronin NB, Dillingham MS, Wigley DBet al., 2014, Structural basis for translocation by AddAB helicase–nuclease and its arrest at χ sites, Nature, Vol: 508, Pages: 416-419, ISSN: 0028-0836

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence χ (Chi)1,2 and is catalysed by either an AddAB- or RecBCD-type helicase–nuclease (reviewed in refs 3, 4). These enzyme complexes unwind and digest the DNA duplex from the broken end until they encounter a χ sequence5, whereupon they produce a 3′ single-stranded DNA tail onto which they initiate loading of the RecA protein6. Consequently, regulation of the AddAB/RecBCD complex by χ is a key control point in DNA repair and other processes involving genetic recombination. Here we report crystal structures of Bacillus subtilis AddAB in complex with different χ-containing DNA substrates either with or without a non-hydrolysable ATP analogue. Comparison of these structures suggests a mechanism for DNA translocation and unwinding, suggests how the enzyme binds specifically to χ sequences, and explains how χ recognition leads to the arrest of AddAB (and RecBCD) translocation that is observed in single-molecule experiments7,8,9.

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Saravanan M, Wuerges J, Bose D, McCormack EA, Cook NJ, Zhang X, Wigley DBet 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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Zhang X, Wigley DB, 2008, The 'glutamate switch' provides a link between ATPase activity and ligand binding in AAA plus proteins, Nature Structural and Molecular Biology, Vol: 15, Pages: 1223-1227, ISSN: 1545-9985

AAA+ proteins carry out diverse functions in cells. In most cases, their ATPase activity is tightly regulated by protein partners and target ligands, but the mechanism for this control has remained unclear. We have identified a conserved link between the ligand binding and ATPase sites in AAA+ proteins. This link, which we call the 'glutamate switch', regulates ATPase activity directly in response to the binding of target ligands by controlling the orientation of the conserved glutamate residue in the DExx motif, switching it between active and inactive conformations. The reasons for this level of control of the ATPase activity are discussed in the context of the biological processes catalyzed by AAA+ proteins.

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