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Journal articleWilkinson M, Troman L, Wan Nur Ismah WA, et al., 2016,
Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones
, eLife, Vol: 5, ISSN: 2050-084XOur 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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Journal articleWillhoft O, Bythell-Douglas R, Mccormack EA, et al., 2016,
Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex
, Nucleic Acids Research, Vol: 44, Pages: 8179-8188, ISSN: 1362-4962We 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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Journal articleZhang N, Darbari VC, Glyde R, et al., 2016,
The bacterial enhancer-dependent RNA polymerase
, Biochemical Journal, Vol: 473, Pages: 3741-3753, ISSN: 1470-8728Transcription 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.
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Journal articleWilkinson M, Chaban Y, Wigley DB, 2016,
Mechanism for nuclease regulation in RecBCD.
, eLife, Vol: 5, ISSN: 2050-084XIn 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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Journal articlevon 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: 2041-1723BRCA2 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.
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Journal articleFilloux A, Freemont P, 2016,
Structural biology: baseplates in contractile machines
, Nature Microbiology, Vol: 1, ISSN: 2058-5276 -
Journal articleChambers S, Kitney R, Freemont P, 2016,
The Foundry: the DNA synthesis and construction Foundry at Imperial College.
, Biochemical Society Transactions, Vol: 44, Pages: 687-688, ISSN: 1470-8752The establishment of a DNA synthesis and construction foundry at Imperial College in London heralds a new chapter in the development of synthetic biology to meet new global challenges. The Foundry employs the latest technology to make the process of engineering biology easier, faster and scalable. The integration of advanced software, automation and analytics allows the rapid design, build and testing of engineered organisms.
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Journal articleZhang 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: 0065-2598Transcription 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.
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Journal articleSawicka 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: 1083-351XThe 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.
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Journal articleZhang 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: 1089-8638In 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.
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