Dr Chris D. Thomas

Profile

Background: Postdoctoral work at the Department of Biochemistry, University of Leicester; Wellcome Career Development Fellowship awarded 1994; moved to University of Leeds in 1995.  Appointed Lecturer in 1998.
Centre Memberships: Antimicrobial Research Centre; Astbury Centre for Structural Molecular Biology.

Research interests

Replication- and conjugation-specific proteins and the maintenance of extrachromosomal DNA
Bacterial resistance to antibiotics is often conferred by the presence of plasmids, or extrachromosomal DNA elements. Small plasmids of Gram positive organisms (such as Staphylococcus aureus) typically replicate via a "rolling circle" mechanism, following cleavage of one DNA strand by a replication initiator protein. The mechanism of this process is related to that used by type-I topoisomerases and other replication initiators such as the gene II protein of bacteriophage M13.
The RepD protein initiates replication of plasmid pC221. We have characterised a new sequence motif at the active site of RepD, enabling it to carry out both DNA cleavage and religation reactions without added ATP. This motif (now recognised by the Pfam designation rep_trans) does not, however, determine the dimerisation properties of the protein.  We have refined the latter via domain swapping and site-directed mutagenesis experiments, and find it located within an unusal asymmetric "tipped" domain within the structure of the protein.
RepD also recruits the PcrA helicase to the origin of replication, which serves to separate the DNA strands permitting replication. This interaction stimulates the processivity of the helicase, enabling a single molecule to unwind the DNA of an entire plasmid.  We are investigating this mechanism using cloned RepD and PcrA studied in vitro using both biochemical and structural techniques, and in a series of collaborations have visualised formation and progression of the RepD:PcrA complex along DNA by AFM and verified that the enhanced processivity is associated with a conformational change in the helicase.
Mobilisation of pC221 has been observed in the presence of self-transmissible plasmids. In addition to the relaxase protein MobA we have identified a crucial accessory protein, MobC, which is common to all plasmids of this family. This work has led to a better understanding of the interactions between MobC and the specific recognition of the cognate transfer origin, oriT, which leads to formation of the functional relaxase complex with MobA; we remain interested in both the regulatory aspects of the interaction and the contributions of the overlapping MobC product.
Bacterial viruses represent another means by which DNA can be transferred from one cell to another.  We are revisiting the replication mechanism of bacteriophage M13 because it bears striking similarities to the replication of pC221 described above, at both enzyme (Gene 2 protein vs. RepD) and DNA (plus-strand origin vs. oriD) levels.  Our goal is to understand the differences between the two model systems which enable pC221 to remain a tighly-regulated replicon of Staphylococcus aureus, in contrast to the effects of M13 infection of Escherichia coli whereby the latter is turned into a factory for production of phage particles.

Qualifications

• BA, MA, Oxford; PhD, Leicester

Student education

Laboratory projects for undergraduate and taught postgraduate students: Typical laboratory projects on offer are concerned with MRSA and DNA replication; more recent projects have also investigated plasmid replication in thermophilic bacteria and the sequence motifs essential for activity of bacteriophage replication.  Projects are usually chosen by students looking to gain experience in molecular biology techniques (PCR, cloning etc.) but the precise content can usually be adjusted to suit the student's interests.  Those with a combined interest in scientific communication and computer animation are also encouraged to discuss opportunities in this area.
Research projects for postgraduate students: Areas of interest include plasmid replication and transfer; maintenance of chromosomal DNA; protein:DNA and protein:protein interactions; and the development of novel targets for antibiotics via these frameworks.  Self-funded students are especially encouraged to apply - please visit the pages of our Graduate School to learn more.  For foundation-year postgraduates interested in rotation projects please contact Dr Thomas to discuss what's new - either by email or (better still) come and knock on my door!
 

Research groups and institutes

• Structural Biology