Target site DNA recognition by Tn3 and Sin resolvases

Holt, Stephanie Elspeth (2014) Target site DNA recognition by Tn3 and Sin resolvases. PhD thesis, University of Glasgow.

Abstract

This work examined the DNA-recognition and synapsis capabilities of Sin and Tn3 resolvases, by alteration of regions of the protein to determine their significance for resolvase function. The importance of the E-helix (an -helical structure in the centre of the resolvase, connecting the catalytic domain and DNA-binding domain of the protein) of Sin recombinase, for DNA recognition at the centre of res site I, and for the formation of a synaptic tetramer was investigated. It was observed that swapping of the E-helix ‘arm’ region from Sin residues to the equivalent residues from Tn3resolvase abolished formation of the synapse. Truncated variants of Sin with the catalytic domain removed, termed ‘tadpoles’, were compared with a full-length activated mutant of Sin (Q115R) to determine the importance of the E-helices in formation of the synapse. From the ‘tadpole’ constructs, it was determined that the E-helix region between residues 109 and 124 is required for the formation of synaptic complexes, as deletion of this region was observed to abolish formation of the complex in vitro. Structure-guided single-residue mutations in this region of the E-helix further demonstrated that residues L112, I116 and M119, which are located on the flat hydrophobic interface between the E-helices of the Sin subunits in the synaptic tetramer complex, have a key role in the formation of this complex. Mutation of any of these residues to an alanine, both in the truncated Sin variants with a wildtype background and in the full-length activated mutant Q115R blocks formation of this complex. The truncated versions of Sin support the hypothesis that a bundle offour E–helices comprises a basic synapsis-competent module. In a related project, transcription activator-like effector recombinases (TALERs), chimeric constructs were constructed with the catalytic domain of an activated variant of Tn3 resolvase
(NM) fused to the DNA binding domain of a TALE protein, TALERs with different N-terminal TALE truncations were constructed. It was demonstrated that the TALER constructs were successfully targeted to designed cognate ‘T-sites’, utilising the DNA-binding specificity of the TALE domain. It was also demonstrated that some of the TALERs constructed are able to catalyse cleavage at T-sites in vitro.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Resolvase, Tn3, Sin.
• Subjects: Q Science > QD Chemistry; Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Stark, Professor W. Marshall
• Date of Award: 2014
• Depositing User: Ms Stephanie Holt
• Unique ID: glathesis:2014-6215
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 31 Mar 2015 08:04
• Last Modified: 03 May 2018 08:15
• URI: https://theses.gla.ac.uk/id/eprint/6215