Dr Gary Sharples

Associate Professor

BSc (Hons) Glasgow, PhD Nottingham

Affiliations
Affiliation	Room number	Telephone
Associate Professor in the Department of Biosciences		+44 (0) 191 33 43986
Fellow of the Wolfson Research Institute for Health and Wellbeing		+44 (0) 191 33 43986

Biography

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, peptoids and chelants 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, ‘bacteria eaters’) 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, peptoids, chelants and other small molecules in partnership with a number of Durham Chemists, including Jas Pal Badyal, Steven Cobb, Karl Coleman, David Hodgson, Ritu Kataky, Matthew, Kitching, Robert Pal, John Sanderson and Gareth Williams.