Prorocic, Marko Milenkov
Sequence selectivity of the resolvase catalytic domain: implications for Z-resolvase design.
PhD thesis, University of Glasgow.
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The extent of sequence specificity of the Tn3 resolvase catalytic domain was investigated by creating libraries of Tn3 site I variants in which all of the central 16 bp were systematically randomised in overlapping 4 bp blocks and recombination deficient and recombination proficient site I variants were selected using two different independent selection strategies employing an activated Tn3 resolvase mutant NM. A degree of flexibility in the sequences permitted in the central 16 bp of the Tn3 site I was observed especially at the positions 4, 7 and 8, but accumulating changes was found to be in general detrimental to recombination. The data was compared to the naturally occurring site I sequences associated with proteins from the Tn3 resolvase family, and integrated with the available structural information revealing a number of residues in the extended arm region that could account for the sequence selectivity observed. The sequence selectivity of the activated Tn3 resolvase NM catalytic domain was tested in the Z-resolvase context employing a similar but less exhaustive selection strategy using a purified Z-resolvase Z-R(NM). Z-resolvases with sequence selectivity that is different to that of Z-R(NM) were constructed using catalytic domains of activated mutants of Sin and Tn21 resolvases and their in vivo and in vitro properties were tested, highlighting the universality of the Z-resolvase approach and its potential for the future applications. A number of issues concerning the Z-resolvase design such as the optimum length of Z-sites, what is the effect of the Zif268 DNA-binding domain on catalytic activity i.e. is it activating or inhibiting, is symmetry a prerequisite in the design of Z-sites or can a Z-resolvase catalyse recombination on sites with an odd number of bases between Zif268 binding sites i.e. one half-site longer than the other, what is the relative influence of the Z-resolvase linker length, and can Z-resolvase be complemented by resolvase and catalyse recombination on appropriately designed hybrid sites were explored. The sequence selectivity of catalytic domains of Sin and Tn21 resolvases was compared using a combination of a mutant library selection strategy and the Sin-Tn21 resolvase hybrid experiments. An attempt to change the sequence selectivity of Tn3 resolvase catalytic domain into that of Sin resolvase, both in the resolvase and Z-resolvase context by mutating the specific residues, implicated in catalytic domain sequence selectivity was performed. The sequence selectivity of activated Tn3 resolvase catalytic domain was successfully changed into that of Sin resolvase
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