Dr Gary Sharples, BSc (Hons) Glasgow, PhD Nottingham
My research interests focus on the molecular mechanisms of genetic recombination, a process that is critical for efficient genome replication, accurate chromosomal repair and generating the exchanges and rearrangements that fuel evolution. Characterisation of these processes has important consequences for our understanding of survival following damage to the hereditary material (allied to research on cancer and ageing) and how new infectious diseases emerge. Much of my work involves structural and functional analyses of enzymes that initiate, process and resolve branched DNA recombination intermediates. Our group is currently studying DNA exchanges occurring at unusually high rates in bacterial viruses and how these genomic rearrangements give rise to multiply drug resistant strains and new pathogenic organisms. We are also testing novel surfaces and peptides for antibacterial activity in an effort to combat increasingly drug-resistant bacterial pathogens.
Lord of the Rings
‘One ring to rule them all, one ring to find them, one ring to bring them all and in the darkness bind them...to DNA’
Bacteriophages or phages (literally, ‘eaters of bacteria’) are widespread viruses infecting virtually every bacterial species. They are the most abundant organisms on Earth and it is estimated that 1025 phages initiate an infection every second. Phages regularly carry genes that can convert relatively benign bacteria into deadly pathogens (e.g. E. coli O157:H7). The lifestyle of phages makes them potent conveyors of genetic information between bacterial species. During the lytic cycle, phage-encoded recombinases promote DNA rearrangements that occasionally result in the acquisition of foreign genes, including virulence determinants. Gene uptake occurs by recombination at sites of limited sequence homology and if the newly assembled combination of genes confers a selective advantage, it will be retained and transmitted to subsequent generations. Our group utilises phage lambda as a model system to study these processes.
Ring One: Exo
Genetic recombination in phage lambda is initiated by the coupled action of Exo and Beta proteins, collectively termed the Red system. Exo is a 26 kDa exonuclease, degrading ssDNA in the 5'-3' direction from a duplex DNA end to produce 3' overhangs. Mononucleotides are released in a highly processive manner at the rate of 10-12 bases per second. The biologically active form of Exo is a trimer arranged as a ring so that a duplex end can be accommodated into the tapering cavity and the exposed ssDNA product is extruded through the central channel. Exo serves as a functional equivalent of RecBCD exonuclease, normally responsible for generating 3' tailed DNA at broken chromosomes in E. coli. RecBCD and a related host nuclease, SbcCD, are disabled by lambda Gam protein to preserve the ends of the phage genome during rolling circle replication.
Ring Two: Beta
Beta protein can generate recombinants by annealing the 3' tailed product generated by Exo to complementary ssDNA sequences. Beta binds to ssDNA, protecting it from attack by single-strand specific nucleases. Beta assembles in solution or in the presence of ssDNA as a multisubunit ring and forms helical filaments on dsDNA which it has annealed. Two pathways of exchange predominate in phage lambda depending on whether a DNA strand is used to invade a homologous duplex or is annealed to a complementary single-strand. The invasion reaction is typical of models for E. coli recombination at a break and requires host RecA to bind single-stranded DNA (ssDNA), locate a homologous duplex and promote strand exchange to create a recombinant joint. The second pathway functions independently of RecA and involves annealing of homologous ssDNA partner sequences by phage Beta protein. More recent studies indicate that exchanges frequently occur within the context of a replication fork.
Ring Three: Orf
Orf participates in the early stages of recombination by apparently supplying a function equivalent to the E. coli RecFOR proteins. These host enzymes assist loading of the RecA strand exchange protein onto ssDNA coated with SSB. The homodimeric Orf protein is arranged as a toroid with a shallow U-shaped cleft, lined with basic residues, running perpendicular to the central cavity. Orf binds DNA, favoring single-stranded over duplex and with no obvious preference for gapped, 3' or 5' tailed substrates. Orf interacts with SSB and both proteins can jointly assemble on ssDNA. How Orf facilitates loading of Beta or RecA proteins is the subject of ongoing research.
Rings and Recombination
Genetic recombination in bacteriophage lambda relies on DNA end processing by Exo to expose 3' tailed strands for annealing and exchange by Beta protein. Beta resembles human Rad52 in which a positively charged groove around the central hub of the ring leaves nucleotide bases accessible for annealing to complementary ssDNA. Exo and Beta do not simply constitute separate steps in initiation of recombination, they physically associate suggesting that degradation and strand annealing reactions are coordinated. Bacterial SSB protein blocks access of recombinases. Orf protein therefore serves to load Beta or RecA onto DNA coated with SSB to promote recombination reactions. This accessory role is conserved throughout biology.
X-philes: RuvC and Rap
Our group has a long-term interest in phage endonucleases which target branched DNA recombination intermediates. Specifically we are studying structure-specific endonucleases from phage lambda (Rap) and phage bIL67 from Lactococcus lactis (RuvC) to explore how 4-way Holliday junctions and 3-way replication forks are distinguished at the molecular level.
We are also interested in novel antimicrobial surfaces, peptides and peptoids in collaboration with Steven Cobb, Karl Coleman and Jas Pal Badyal in Chemistry.
School of Biological and Biomedical Sciences
- Bacteriophage genome rearrangements
- Evolution of bacterial pathogenicity
- Horizontal gene transfer
- Mechanisms of homologous recombination
- Antimicrobial surfaces and peptides
- Sharples, G.J. (2001). The X philes: structure-specific endonucleases that resolve Holliday junctions. Mol Microbiol 39: 823-834.
- Sharples, G.J., Ingleston, S.M. & Lloyd, R.G. (1999). Holliday junction processing in bacteria: insights from the evolutionary conservation of RuvABC, RecG, and RusA. J Bacteriol 181: 5543-5550.
Journal papers: academic
- Green, V., Curtis, F.A., Sedelnikova, S., Rafferty, J.B. & Sharples, G. (2013). Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry. Molecular Microbiology 89(6): 1240–1258.
- Matsubara, K., Malay, A.D., Curtis, F.A., Sharples, G.J. & Heddle, J.G. (2013). Structural and functional characterization of the Redβ recombinase from bacteriophage λ. PLOS ONE 8(11): e78869.
- Wood, T.J., Hurst, G.A., Schofield, W.C.E., Thompson, R.L., Oswald, G., Evans, J.S.O., Sharples, G.J., Pearson, C., Petty, M.C. & Badyal, J.P.S. (2012). Electroless deposition of multi-functional zinc oxide surfaces displaying photoconductive, superhydrophobic, photowetting, and antibacterial properties. Journal of Materials Chemistry 22(9): 3859-3867.
- Curtis, F.A., Reed, P., Wilson, L.A., Bowers, L.Y., Yeo, R.P., Sanderson, J.M., Walmsley, A.R. & Sharples, G.J. (2011). The C-terminus of the phage λ Orf recombinase is involved in DNA binding. Journal of Molecular Recognition 24(2): 333-340.
- Sharples, G.J. (2009). For absent friends: life without recombination in mutualistic gamma-proteobacteria. Trends in Microbiology 17(6): 233-242.
- Carrasco, B., Cañas, C., Sharples, G.J., Alonso, J.C. & Ayora, S. (2009). The N-terminal region of the RecU Holliday junction resolvase is essential for homologous recombination. Journal of Molecular Biology 390(1): 1-9.
- Pan, P.-S., Curtis, F.A., Carroll, C.L., Medina, I., Rodrigeuz, R., Liotta, L.A., Sharples, G.J. & McAlpine, S.R. (2006). Novel antibiotics: C-2 symmetrical macrocycles inhibiting Holliday junction DNA binding by E. coli RuvC. Bioorg Med Chem 14: 4731-4739.
- Wen, Q., Mahdi, A.A., Briggs, G.S., Sharples, G.J. & Lloyd, R.G. (2005). Conservation of RecG activity from pathogens to hyperthermophiles. DNA Repair - Amst 4: 23-31.
- Curtis, F.A., Reed, P. & Sharples, G.J (2005). Evolution of a phage RuvC endonuclease for resolution of both Holliday and branched DNA junctions. Molecular Microbiology 55(5): 1332-1345.
- Maxwell, K. L., Reed, P., Zhang, R. G., Beasley, S., Walmsley, A. R., Curtis, F. A., Joachimiak, A., Edwards, A. M. & Sharples, G. J. (2005). Functional similarities between phage lambda Orf and Escherichia coli RecFOR in initiation of genetic exchange. Proceedings of the National Academy of Sciences of the United States of America 102(32): 11260-11265.
- Sanchez, H., Kidane, D., Reed, P., Curtis, F.A., Cozar, M.C., Graumann, P.L., Sharples, G.J. & Alonso, J.C. (2005). The RuvAB branch migration translocase and RecU Holliday junction resolvase are required for double-stranded DNA break repair in Bacillus subtilis. Genetics 171: 873-883.
- Borges-Walmsley, M.I., Du, D., McKeegan, K.S., Sharples, G.J. & Walmsley, A.R. (2005). VceR regulates the vceCAB drug efflux pump operon of Vibrio cholerae by alternating between mutually exclusive conformations that bind either drugs or promoter DNA. J Mol Biol 349(2): 387-400.
- Moore, T., Sharples, G.J. & Lloyd, R.G. (2004). DNA binding by the meningococcal RdgC protein associated with pilin antigenic variation. J Bacteriol 186: 870-874.
- Sharples, G. J., Curtis, F. A., McGlynn, P. & Bolt, E. L. (2004). Holliday Junction Binding and Resolution by the Rap Structure-specific Endonuclease of Phage lambda. Journal of Molecular Biology 340(4): 739-751.
- Liotta, L.A., Medina, I., Robinson, J.L., Carroll, C.L., Pan, P.-S., Corral, R., Cook, K.M., Johnston, J.V.C., Curtis, F.A., Sharples, G.J. & McAlpine, S.R. (2004). Novel antibiotics: second generation macrocyclic peptides designed to trap Holliday junctions. Tet Lett 45: 8447-8450.
- Loughlin, M.F., Barnard, F.M., Jenkins, D., Sharples, G.J. & Jenks, P.J. (2003). Helicobacter pylori mutants defective in RuvC Holliday junction resolvase display reduced macrophage survival and spontaneous clearance from the murine gastric mucosa. Infect Immun 71: 2022-2031.
- Mahdi, A.A., Briggs, G.S., Sharples, G.J., Wen, Q. & Lloyd, R.G. (2003). A model for dsDNA translocation revealed by a structural motif common to RecG and Mfd proteins. EMBO J 22: 724-734.
- Moore, T., McGlynn, P., Ngo, H.P., Sharples, G.J. & Lloyd, R.G. (2003). The RdgC protein of Escherichia coli binds DNA and counters a toxic effect of RecFOR in strains lacking the replication restart protein PriA. EMBO J 22: 735-745.
- Rafferty, J.B., Bolt, E.L., Muranova, T.A., Sedelnikova, S.E., Leonard, P., Pasquo, A., Baker, P.J., Rice, D.W., Sharples, G.J. & Lloyd, R.G. (2003). The structure of Escherichia coli RusA endonuclease reveals a new Holliday junction DNA binding fold. Structure 11: 1557-1567.
- Ingleston, S.M., Dickman, M.J., Grasby, J.A., Hornby, D.P., Sharples, G.J. & Lloyd, R.G. (2002). Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae. Eur J Biochem 269: 1525-1533.
- Sharples, G. J., Bolt, E. L. & Lloyd, R. G. (2002). RusA proteins from the extreme thermophile Aquifex aeolicus and lactococcal phage r1t resolve Holliday junctions. Molecular Microbiology 44(2): 549-559.
- Bolt, E.L., Lloyd, R.G. & Sharples, G.J. (2001). Genetic analysis of an archaeal Holliday junction resolvase in Escherichia coli. J Mol Biol 310(3): 577-589.
- Bolt, E.L., Sharples, G.J. & Lloyd, R.G. (2000). Analysis of conserved basic residues associated with DNA binding (Arg69) and catalysis (Lys76) by the RusA Holliday junction resolvase. J Mol Biol 304: 165-176.
- Ingleston, S.M., Sharples, G.J. & Lloyd, R.G. (2000). The acidic pin of RuvA modulates Holliday junction binding and processing by the RuvABC resolvasome. EMBO J 19: 6266-6274.
- Sharples, G.J., Corbett, L.M., McGlynn, P. & Bolt, E.L. (1999). DNA structure specificity of Rap endonuclease. Nucl Acids Res 27: 4121-4127.
- Bolt, E.L., Sharples, G.J. & Lloyd, R.G. (1999). Identification of three aspartic acid residues essential for catalysis by the RusA Holliday junction resolvase. J Mol Biol 286: 403-415.
- Sharples, G.J., Corbett, L.M. & Graham, I.R. (1998). Lambda Rap protein is a structure-specific endonuclease involved in phage recombination. Proc Natl Acad Sci USA 95: 13507-13512.