Dr Chris Thomas

Dr Thomas obtained his B.A. and M.A. at Hertford College, Oxford in 1984, and his Ph.D. in Biochemistry from the University of Leicester in 1988. Following postdoctoral work at Leicester he was awarded a Wellcome Trust Career Development Fellowship in 1994, and joined the School of Biochemistry & Molecular Biology at the University of Leeds in 1995. Dr Thomas was appointed to a Lectureship in Biochemistry in 1998.

Research Areas: Plasmid Biology; Protein-DNA and Protein-Protein Interactions

Current Research Projects:

Replication-specific proteins and the maintenance of extrachromosomal DNA

Bacterial resistance to antibiotics is often conferred by the presence of plasmids, or extrachromosomal DNA elements. It is now apparent that many small plasmids of Gram positive organisms (such as Staphylococcus aureus) replicate via a ?rolling circle? mechanism, following the cleavage of one of the DNA strands by a replication initiator protein. Our primary interest concerns how these proteins work. One such example is RepD, from staphylococcal plasmid pC221.

Through the interaction of RepD with a unique specificity site at the double-stranded replication origin we are able to study sequence-specific protein:DNA binding. We are also investigating DNA structure, whereby a subsequent conformational change at the replication origin permits the protein to nick the DNA at the adjacent nick site. RepD becomes covalently attached to the 5' end of this nick; the free 3'OH serves to prime replication of a new (+) strand in vivo or is utilised in a religation reaction by RepD in vitro. Termination of replication also requires the covalent RepD-DNA complex to correctly identify the nick site, influenced by a novel DNA:DNA interaction.

RepD has also been found to alter the behaviour of the PcrA helicase, required to separate the strands of plasmid DNA following the initation of replication by RepD. We have created a model for the initiation complex formed between RepD, PcrA and origin DNA in vitro. RepD enhances the processivity of PcrA, making it more suited to its role in plasmid DNA replication. In addition to this interaction between between different proteins, we have also characterised the interactions between monomers of Rep protein variants which has identified specificity determinants governing formation of the Rep dimer.

We are using site-directed mutagenesis of both RepD and its DNA target to investigate the catalytic mechanism of the protein as a model for other type-I topoisomerases, and studying the interactions with oligonucleotide and plasmid substrates to mimic the events of initiation and termination of replication. In collaboration with Professor Simon Phillips we are well advanced in solving the structure of RepD by X-ray diffraction methods.

Plasmid mobilisation and conjugative DNA transfer

The transfer of DNA between bacteria results in genetic diversity, often with important medical consequences as strains resistant to multiple antibiotics emerge. The transfer mechanism involves a rolling circle process; we are studying in the mobilisation functions of plasmid pC221 as a simple system embodying the initial events of conjugative transfer in Staphylococcus aureus.

The MobA transesterase requires the presence of an accessory protein, MobC, for both efficient mobilisation in vivo and formations of a relaxosome complex in vitro. We have characterised the protein:DNA interactions between MobC and the origin of transfer: curiously it is the MobA protein which disciminates between the origin sequences of related plasmids. Having defined a minimal function origin we are currently mapping the region of DNA sequence specificity within MobA.

Related nicking-closing processes

The Mob and Rep proteins of pC221 have different amino acid motifs surrounding tyrosine nucleophile at the active site. While these motifs are well characterised for both the Mob relaxases and many replicons unrelated to pC221, the RepD motif is currently poorly understood. We are therefore studying proteins which possess this motif from a variety of sources: this includes the specific initiator protein gpII from bacteriophage M13, in addition to replication-related proteins isolated from plasmids of other Gram-positives and potential relaxases encoded within conjugative transposons.

Type-II topoisomerases

The topoisomerases also display phosphodiester bond cleavage and religation, although without the complexity of sequence specificity required by the Mob and Rep proteins. The Type-II enzymes also cleave both strands at their target: DNA gyrase then introducing negative supercoils while Topoisomerase IV plays a role in the decatenation of DNA.

Using the kinetic approaches derived from the study of RepD and MobA we have characterised the topoisomerase activities of DNA gyrase and Topoisomerase IV of Staphylococcus aureus, and the associted susceptibilities to antibiotics of the quinoloine family. Our structural studies in this area have again yielded crystals suitable for structure determination by X-ray diffraction methods.