University of Edinburgh
Bower, Edward Kenneth Merrick.
The evolution of restriction-modification systems.
Degree: PhD, 2017, University of Edinburgh
Restriction Modification (R-M) systems prevent the invasion of foreign genetic material into bacterial cells and are therefore important in maintaining the integrity of the host genome. The spread of antibiotic resistance, which is proposed to occur via the transfer of foreign genes to the bacterial genome, makes the subject of R-M systems extremely relevant. R-M systems are currently classified into four types (I to IV) on the basis of differences in composition, target recognition, cofactors and the manner in which they cleave DNA. Kennaway et al (2012) proposed that there is an evolutionary link between Types I and II. Comparing the structures of examples from two of the subfamilies of Type II systems (IIB and IIG) to those of Type I structures, similarities can be observed. Due to the fact that Type II R-M systems cut DNA at fixed positions, they can be used to obtain genetic material selectively. They have therefore proven to be invaluable in molecular biology. One aspect of this project aims to create a novel R-M system, a pseudo-Type II system, by removing the molecular motors from the restriction subunit of a Type I system and fusing the remaining nuclease domain to a known Type I methyltransferase (MTase). This will not only provide evidence to support the theory that evolution has produced a pared down form of the Type I systems in the Type II systems, but it may also become a useful biological tool. This thesis describes the several attempts at doing this and how the subsequent constructs were expressed, purified and assayed to varying degrees of success. An important characteristic of the Type I systems is their ability to methylate DNA, and it is the mechanism via which host DNA is protected from restriction. This is another subject investigated in this project. As with the nuclease activity of the Type I systems, the site at which DNA is methylated is dictated by the HsdS subunit. It is described here how this subunit can be altered to change the sequence of DNA that is recognised by the system. Again, using Type II system subtypes as a reference, various mutations were made to the HsdS subunit of an MTase from Staphylococcus aureus. This is in an effort to bring about a new mode of action, but also to provide further evidence for an evolutionary link between the two system types. The HsdM and HsdS subunits are expressed from two separate genes at the same locus. There is a frameshift between the genes where the start of the hsdS gene occurs a few base pairs upstream from the stop codon of the hsdM gene. This work shows that removing this frameshift creates an MS fusion product, and in vivo studies show that this product has methylase activity and can form an active restriction complex when the HsdR subunit is added. The product can also be over-expressed and purified, and shows in vitro restriction activity on addition of the HsdR subunit protein. The HsdS subunit is composed of two target recognition domains (TRDs), each dictating one part of the bipartite recognition sequence. These TRDs can be…
Subjects/Keywords: restriction enzymes; estriction-modification; methylation; R-M systems; Type II R-M systems
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APA (6th Edition):
Bower, E. K. M. (2017). The evolution of restriction-modification systems. (Doctoral Dissertation). University of Edinburgh. Retrieved from http://hdl.handle.net/1842/29528
Chicago Manual of Style (16th Edition):
Bower, Edward Kenneth Merrick. “The evolution of restriction-modification systems.” 2017. Doctoral Dissertation, University of Edinburgh. Accessed December 06, 2019.
MLA Handbook (7th Edition):
Bower, Edward Kenneth Merrick. “The evolution of restriction-modification systems.” 2017. Web. 06 Dec 2019.
Bower EKM. The evolution of restriction-modification systems. [Internet] [Doctoral dissertation]. University of Edinburgh; 2017. [cited 2019 Dec 06].
Available from: http://hdl.handle.net/1842/29528.
Council of Science Editors:
Bower EKM. The evolution of restriction-modification systems. [Doctoral Dissertation]. University of Edinburgh; 2017. Available from: http://hdl.handle.net/1842/29528