DNA ligases play a critical role in DNA replication, repair and recombination in all living organisms, joining DNA ends whenever new DNA is made or when damaged DNA is repaired. Bacterial ligases use NAD+ as their energy source, whereas eukaryotic ligases use ATP. This difference allows selective targeting of bacterial NAD+-dependent ligases for antibacterial therapies.
Recently we solved the X-ray crystal structure of an NAD+-dependent DNA ligase from a T5-like bacteriophage in a new DNA-bound state. This revealed a potentially important novel role for the DNA ligase BRCT domain, which is not present in ATP-dependent DNA ligases and which has not previously been visible in NAD+-dependent DNA ligase structures.
This PhD project will focus on establishing, using structural and molecular approaches, the molecular function of the BRCT domain in NAD+-dependent DNA ligases, with a view to exploring if this is a valid target for novel antimicrobial therapies. The objectives are:
(1) To test hypotheses about the function of the BRCT domain. Molecular genetic tools, in vitro assays and cell-based protocols will be used to analyse the molecular and cellular function of the BRCT domain of NAD+-dependent DNA ligases via reverse genetic approaches. Structure-based design of mutations will allow investigation of the roles of specific ligase amino acids.
(2) To establish if the novel structural role of the bacteriophage ligase BRCT domain is conserved across ligases from evolutionarily divergent species. Crystal structures of cellular DNA ligase(s) with DNA will be determined in the same way that we captured the bacteriophage ligase-DNA complex. A broad range of NAD+-dependent DNA ligases from medically relevant (e.g. Vibrio, Clostridium, Salmonella, Streptococcus, MRSA) and evolutionarily diverse bacteria and archaea (e.g. Haloferax, which acquired the ligase by horizontal gene transfer from bacteria) will be systematically screened to find soluble and active enzymes that are amenable to structural characterisation by X-ray crystallography.
(3) To combine bioinformatics analysis of a diverse range of NAD+-dependent DNA ligase sequences with the functional and structural analyses to determine the function of the BRCT domain and whether this function offers a viable target for antimicrobial drug development.
We will use complementary structural, biochemical and bioinformatics approaches, along with in vitro and in vivo assays. The student will develop a broad range of skills in structural biology and biochemistry and in the molecular microbiology and molecular genetics of DNA ligases.
https://www.ed.ac.uk/profile/dr-julia-richardson
https://macneill.wp.st-andrews.ac.uk
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