... understanding life in molecular detail

Dr Chris Thomas

plasmid, replication, mobilisation, relaxase


We are interested in the mechanisms by which DNA is replicated, maintained and transmitted among pathogens such as Staphylococcus aureus.  Our focus is on the enzymology of rolling-circle replication, with a strong emphasis on the structure-function relationships of protein-DNA interactions typified by the initiator RepD of pC221, as well as the stimulation by RepD of the essential helicase PcrA.

Current major projects include:
  • Structural studies of RepD and RepSTK1
  • DNA recognition and cleavage by relaxases of the rep_trans family
  • Protein:protein interactions and stimulation of PcrA helicase activity
  • Dimerisation specificity and evolution of new incompatibility groups

Activities of replication initiator proteins

Plasmids of the staphylococcal pT181 family replicate via a rolling circle mechanism.  Replication is initiated by plasmid-specified Rep proteins, such as the RepD protein encoded by the chloramphenicol-resistance plasmid pC221.  The active site of RepD is defined by sequences which make up the Rep_trans motif (pfam 02486), which is unrelated to the previously-characterised His-hydrophobic-His motif also widespread among rolling circle replicons.

In collaboration with Professor Simon Phillips we have recently obtained the crystal structures defining two diverse Rep_trans relaxases: derivatives of RepD, RepC, RepE and RepN (specified by plasmids pC21, pT181, pS194 and pCW7) as examples of initiators from plasmids of the pathogen Staphylococcus aureus, and an active fragment of the Rep protein of the cryptic plasmid pSTK1 obtained from the moderate thermophile Geobacillus stearothermophilus.  Despite less than 13% sequence identity overall these structures are remarkably similar, and present the active site in adjacent strands of a beta sheet which may serve to encircle DNA.

Fig. 1Fig. 2

Stimulation of PcrA helicase by Rep proteins

PcrA is an essential helicase of Gram-positive organisms such as Staphylococcus aureus and Bacillus subtilis.  Through collaborations with Neil Thomson (Astbury Centre), Panos Soultanas (Nottingham) and Martin Webb (Mill Hill) we have shown that RepD is able to recruit PcrA to the plasmid replication origin, oriD, as well as convert PcrA from a poor to a highly-processive enzyme capable of unwinding an entire plasmid.  We are currently working with Colin Fishwick (Chemistry) to study novel inhibitors of PcrA which may be of use both in mechanistic studies of the Rep:PcrA interaction and in the development of new classes of antimicrobials.

Structural studies of type-II topoisomerases

Also in collaboration with Professor Simon Phillips we have previously obtained structural data for the catalytic subunit of staphylococcal topoisomerase IV.  This is an established target for existing antibiotics, and also possesses DNA cleavage-religation activity.  Like the Rep_trans motif, topoisomerases of this family use a series of acidic residues to coordinate a divalent metal ion at the catalytic centre, but the structural fo9lds used to achieve this are unrelated between topoisomerases and Rep proteins.

Defining the components of plasmid mobilisation

We have also worked on the mobilisation properties of pC221, whereby it can be transferred between cells by conjugation.  The MobA protein is a relaxase of the His-hydrophobic-His family; we also identified an accessory protein (MobC) essential for the transfer process and defined the unusual arrangement of direct repeats bound by MobC which are essential for cleavage at the origin of transfer, oriT.

Detailed research programme                  Close ▲
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Lecturer (Leeds) 1998-present
MA (Oxford) PhD (Leicester)


Postdoctoral researcher (Leicester) 1988-94
Wellcome Trust Research Career Development Fellow (Leicester) 1994-95
Wellcome Trust Research Career Development Fellow (Leeds) 1995-98

Astbury 6.02
School of Molecular and Cellular Biology
0113 343 3040
bmbcdt@leeds.ac.uk
www.fbs.leeds.ac.uk/plasmids

Selected Publications

  1. Carr, S.B., Mecia, L.B., Phillips, S.E.V. and Thomas, C.D. (2013) Identification, characterization and preliminary X-ray diffraction analysis of the rolling-circle replication initiator protein from plasmid pSTK1. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 69 (10):1123-1126. doi:10.1107/S1744309113023828

  2. Phillips, S.E.V., Carr, S.B., Mecia, L.B., Stelfox, A.J. and Thomas, C.D. (2013) Structural Studies of Rolling Circle Replication Initiator Proteins. Biophys. J. 104 (2 Suppl. 1): 73a-74a.doi:10.1016/j.bpj.2012.11.444

  3. Arslan, S., Khafizov, R., Thomas, C.D., Chemla, Y.R. and Ha, T. (2015) Engineering of a superhelicase through conformational control. Science 348(6232):344-347. doi:10.1126/science.aaa0445

  4. Carr, S.B., Phillips, S.E.V. and Thomas, C.D. (2016) Structures of replication initiation proteins from staphylococcal antibiotic resistance plasmids reveal protein asymmetry and flexibility are necessary for replication. Nucleic Acids Res. 44(5):2417-2428. doi:10.1093/nar/gkv1539