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Title: The evolution of restriction-modification systems
Author: Bower, Edward Kenneth Merrick
ISNI:       0000 0004 7225 3090
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2017
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Abstract:
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 altered, bringing about a change in the sequence of DNA recognised by the enzyme. In this thesis, it is shown that the C-terminal TRD can be removed and that the subsequent “Half S” enzyme possesses both methylase and restriction activity in vivo and that its recognition sequence is different from that of the wild-type enzyme. After the successful creation of both “MS fusion” and “Half S” recombinant proteins of the Sau1, Type I system from a CC398 strain of Staphylococcus aureus, a further construct was produced. This possesses both in vivo and in vitro activity. The novel “M Half S Fusion” enzyme not only links the two aspects of this project but also creates a structure similar to some seen in the Type II systems. This shows that the Type I systems can be manipulated to change their mode of action but also supports the idea that Types I and II are evolutionarily linked. By making the alterations in a step-wise fashion identifies that these structural changes can create viable enzymes, and that they could have occurred through the process of evolution.
Supervisor: Mowat, Christopher ; Campopiano, Dominic Sponsor: Engineering and Physical Sciences Research Council (EPSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.739086  DOI: Not available
Keywords: restriction enzymes ; estriction-modification ; methylation ; R-M systems ; Type II R-M systems
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