Tn3 resolvase-catalysed recombination: Assembly and activation of the site I synapse

Olorunniji, Femi John (2006) Tn3 resolvase-catalysed recombination: Assembly and activation of the site I synapse. PhD thesis, University of Glasgow.

Abstract

The assembly and catalytic activation of the site I synapse in resolvase-catalysed recombination was investigated by a combination of mutagenesis and chemical modification of DNA substrates. The binding and catalytic properties of activated Tn3 resolvase mutants were compared with those of wild-type resolvase with the aim of understanding how activating mutations deregulate site I synapsis and catalysis. The findings indicate that structural changes in the resolvase-site I dimer complex are important for the formation of a stable site I synapse. Efficient catalysis of site I x site I recombination by activated resolvase mutants is highly correlated with formation of stable site I synapse in polyacrylamide bandshift assays. Conserved residues that are presumed to form the active site of resolvase were mutated in activated mutants of Tn3 resolvase, and the binding, synapsis and catalytic properties of the mutant resolvases were characterised. Certain mutations in putative active site residues of resolvase resulted in decrease in the yield and/or stability of the site I synapse, while mutations in another set of residues in the putative active site destabilised the dimer complex and stabilised the site I synapse. These results suggest a role for the active site in site I synapsis. Methylphosphonate replacements of phosphodiester groups close to the scissile position in site I show that direct contact of resolvase with the centre of site I is important for synapsis, but not necessarily for binding. Such contacts are not seen in the published structures of the site 1 synapse. The apparent lack of contact between catalytic residues and the DNA in cocrystal resolvase structures suggests that the engagement of catalytic residues with the substrate is at the point of catalytic activation. The results of site I cleavage experiments with resolvase mutants show that while several conserved residues are important for the recombination activity of resolvase, only a few of these are essential for the activation of the catalytic process. The effects of methylphosphonate substitution at the phosphate immediately 3' to the scissile position on site I cleavage suggests a catalytic role for this phosphate, such as recruitment of positively charged catalytic residues into the active site prior to catalysis.

• Item Type: Thesis (PhD)
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Stark, Prof. Marshall
• Date of Award: 2006
• Depositing User: Enlighten Team
• Unique ID: glathesis:2006-82029
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 23 Feb 2021 14:00
• Last Modified: 28 Nov 2022 14:11
• Thesis DOI: 10.5525/gla.thesis.82029
• URI: https://theses.gla.ac.uk/id/eprint/82029