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Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond	Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones Martin Wilkinson , Lucy Troman , Wan Ak Wan Nur Ismah , Yuriy Chaban , Matthew B. Avison , Mark S. Dillingham , Dale B. Wigley Elife 5 DOI: 10.7554/eLife.22963 Open Access Show Abstract ▼ Abstract: 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.	Dec 2016
I04-Macromolecular Crystallography	Structural basis for translocation by AddAB helicase-nuclease and its arrest at χ sites Wojciech Krajewski , Xin Fu , Martin Wilkinson , Nora Cronin , Mark S. Dillingham , Dale B. Wigley Nature DOI: 10.1038/nature13037 PMID: 24670664 Diamond Proposal Number(s): [8015] Show Abstract ▼ Abstract: 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 experiments	Mar 2014
I02-Macromolecular Crystallography	Insights into Chi recognition from the structure of an AddAB-type helicase-nuclease complex Saikrishnan Kayarat , Joseph T. Yeeles , Neville S. Gilhooly , Wojciech W. Krajewski , Mark S. Dillingham , Dale B. Wigley The Embo Journal 31 (6), 1568 - 1578 DOI: 10.1038/emboj.2012.9 PMID: 22307084 Diamond Proposal Number(s): [6385] Open Access Show Abstract ▼ Abstract: In bacterial cells, processing of double?stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence Chi and is catalysed by either an AddAB? or RecBCD?type helicase–nuclease. Here, we report the crystal structure of AddAB bound to DNA. The structure allows identification of a putative Chi?recognition site in an inactivated helicase domain of the AddB subunit. By generating mutant protein complexes that do not respond to Chi, we show that residues responsible for Chi recognition are located in positions equivalent to the signature motifs of a conventional helicase. Comparison with the related RecBCD complex, which recognizes a different Chi sequence, provides further insight into the structural basis for sequence?specific ssDNA recognition. The structure suggests a simple mechanism for DNA break processing, explains how AddAB and RecBCD can accomplish the same overall reaction with different sets of functional modules and reveals details of the role of an Fe–S cluster in protein stability and DNA binding.	Feb 2012

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