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Title: Characterisation of the streptococcal DNA polymerase I flap endonuclease domain
Author: Lau, Roxanne
ISNI:       0000 0004 7226 6350
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2018
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The rise of antibiotic-resistant bacteria has quickly become one of the most concerning threats to confront modern medicine. This project focuses on the bacterial pathogen Streptococcus pneumoniae in particular, resistant strains of which play a role in the deaths of some 7,000 people annually, in the USA alone. It is apparent that new strategies are urgently needed to combat the growing antibiotic-resistance problem. One such strategy might be to look for novel drug targets in the underexplored area of the bacterial DNA replication machinery. Here, we report the biochemical and structural characterization of the streptococcal FEN domain (SpFEN), a crucial player in DNA replication, as a potential novel target for future rational drug design. Comparison of biochemical data between the WT SpFEN and four active-site mutants reveal conserved aspartate residues that are required for both exo- and endonuclease activities. Structural data was also obtained for SpFEN, providing some of the first structures of FENs from pathogenic organisms. The SpFEN apo structure as well as two co-crystal structures of the SpFEN:DNA complex have been determined to high resolutions (1.78 Å, 1.65 Å, and 2.13 Å respectively) by X-ray crystallography, revealing different conformations of the domain. In addition, the structure of the streptococcal full-length DNA polymerase is presented here at 4.01 Å resolution. This full-length structure was also solved in complex with DNA, which was found to bind to the polymerase domain of the protein. The structure also reveals a 23.1° rotation of the FEN domain relative to the Klenow fragment when compared with the Taq polymerase structure. Lastly, FRET-based biochemical screening of SpFEN against a 1000 "fragment" library identified several possible inhibitors demonstrating the feasibility of inhibitor discovery through high throughput screening approaches.
Supervisor: Sayers, Jon ; Baker, Patrick Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available