Untangling E. coli Xer recombination through Atomic Force Microscopy

Provan, James Iain (2021) Untangling E. coli Xer recombination through Atomic Force Microscopy. PhD thesis, University of Glasgow.

Due to Embargo and/or Third Party Copyright restrictions, this thesis is not available in this service.

Abstract

The topology of DNA is crucial to cellular processes such as replication, transcription, and recombination. The protein-DNA interactions of these processes can induce major topological changes into DNA molecules. The Xer site-specific DNA recombination system is a very well characterised topology-dependent mechanism. First discovered in Escherichia coli, Xer consists of a heterotetrametric tyrosine recombinase complex paired with several accessory proteins. Xer is involved in the resolution of chromosome dimers, and in the conversion of supercoiled plasmid multimers to monomeric states. Both Xer-dependent mechanisms are required for equal and faithful segregation of E. coli DNA at cell division.

There has long been an aim to observe the topological complexity of DNA, e.g., the products of site-specific recombination, on a single molecule level. Existing techniques such as rotary shadowing electron microscopy have seen little recent development and have significant practical limitations. In this thesis I demonstrate the application of high-resolution Atomic Force Microscopy (AFM) in liquid to the topological determination of DNA knots and catenanes. The E. coli Xer synapse was used as a tool to generate knotted or catenated DNA in vitro. Several aspects of knot and catenane generation e.g., reaction conditions, substrate length modifications, repeatability, and DNA purification were probed in depth.

Several putative DNA knots were classified from AFM micrographs by tracing their path and assigning chirality based on the topographical profile of the DNA-DNA crossovers. Every possible knotted configuration of three AFM-traced DNA molecules was determined by hand. These were most likely 4-node knot (4-1), 5-1* twist-knot, and an unknot. Subsequently, a 4-node catenated DNA with a homogenous topological nature was studied by AFM to focus solely on the discrimination of over-crossing DNA strands versus under-crossing strands. Individually, several AFM micrographs of 4-node catenanes were of extremely high image quality, where their crossovers could be readily determined and were consistent with prior biochemical evidence of chirality. However, the majority of catenanes adopted configurations where their crossings were clustered indecipherably. Only 13% of the traced 4-node catenanes were of conformations that might be conducive to the determination of crossover directionality.

The work described in this thesis represents the first topological study of DNA knots and catenanes by AFM, and demonstrated the utility of this technique. Future studies of DNA knots and catenanes by AFM will require further optimisation of sample deposition conditions and imaging parameters to reduce the tendency of DNA molecules to cluster together, and to emphasise the directionality of knotted or catenated DNA crossovers.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: DNA, molecular biology, biochemistry, topology, recombination.
Subjects: Q Science > Q Science (General)
Q Science > QH Natural history > QH345 Biochemistry
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Supervisor's Name: Colloms, Dr Sean
Date of Award: 2021
Embargo Date: 28 January 2024
Depositing User: Dr James Iain Provan
Unique ID: glathesis:2021-81962
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 29 Jan 2021 09:14
Last Modified: 01 Jun 2021 17:18
Thesis DOI: 10.5525/gla.thesis.81962
URI: http://theses.gla.ac.uk/id/eprint/81962

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