Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796826
Title: Binding of Tn3 resolvase to its resolution site
Author: Blake, David Gordon
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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Abstract:
Tn3 resolvase binds to three subsites within its DNA target, res. Complexes of resolvase bound to res can be isolated using non-denaturing polyacrylamide electrophoresis (PAGE). Six complexes are generated with Tn3 res and resolvase, but only three complexes have been reported for the related gammadelta resolution system. By varying the conditions used for non-denaturing PAGE it was demonstrated that the different methods used were not solely responsible for the observed differences in the patterns of complexes produced. In vivo overexpression of Tn3 resolvase in the presence of several tritiated amino acids was used to produce tritiated resolvase. Substantial purification of the radiolabelled resolvase was achieved using a method adapted for use with small amounts of material, but based on a previously published resolvase purification method. The 3H-resolvase sample produced was used, with 32P end-labelled DNA fragments, to determine the relative and absolute stoichiometry of the protein/DNA complexes observed for Tn3. Although with res one complex (complex 5) was not isolated, the observed results indicate that the stoichiometry of the least retarded complex (complex 1) is one resolvase monomer per res site, and that this value increases by one monomer per res for each successively more retarded complex, to a value of six resolvase monomers per res site for the most retarded complex (complex 6). This is consistent with the prediction that each subsite of res can be bound by a dimer of resolvase. A synthetic DNA was used to generate subsite II in isolation from subsites I and III. Binding of Tn3 resolvase to subsite II produced two (or sometimes three) complexes. The stoichiometry of complexes of subsite II with resolvase was consistent with results produced for res, i.e. the first complex had a ratio of one monomer of resolvase per DNA site and this value increased by one monomer per site for successively more retarded complexes. Synthetic derivatives of a subsite II sequence were also produced, with an additional 5 or 10 base pairs (bp) of DNA sequence inserted at the subsite centre (named subsite (II + 5) and subsite (II + 10), respectively). Tn3 resolvase was shown to bind to these altered subsites, but the distribution of complexes produced was different from those for subsite II. Complexes of subsite (II + 10) had the same stoichiometry as equivalent complexes of subsite II, as did the first two complexes of subsite (II + 5). DNase I footprinting and methylation interference analysis of Tn3 resolvase binding to subsites II, (II + 5) and (11 + 10) suggested that the protein/DNA contacts made were equivalent to those made by resolvase binding to a normal subsite within res. Hence it was concluded that the differences in complex formation observed with subsites (II + 5) and (II + 10) are likely to result from an alteration of contacts made between resolvase molecules bound at these sites. Binding analysis of several different DNA fragments carrying subsite II or subsite (II + 10) was used to show that binding of a monomer or a dimer of Tn3 resolvase induces a bend at the centre of each subsite. From previous studies it seems likely that resolvase monomer-induced bending of DNA is a common feature for binding to each subsite of res. Purified samples of gammadelta resolvase and of a gammadelta resolvase mutant, gammadelta M106C, (both supplied by Nigel Grindley) were also used in binding experiments. The amino acid substitution present in gammadelta M106C allows the formation of covalently linked dimers of resolvase. Comparison of binding of Tn3, gammadelta and gammadelta M106C resol vases to subsites II, (II + 5) and (II + 10) showed that gammadelta resolvase binds as a predominantly dimeric species, and that Tn3 resolvase binds as a predominantly monomeric species. This comparison also suggested that binding of Tn3 resolvase to subsite II is co-operative. A model of Tn3 resolvase binding to res, based on these results, is proposed, in which the majority of resolvase is monomeric and occupies the res site in six successive steps, each representing binding of a resolvase monomer to one half of a subsite, until each subsite is bound by a dimer of resolvase. The subsites are not occupied in only one specific order, but in a variable order, probably reflecting the affinity of resolvase for each target site. It seems likely that resolvase bound to one half of a subsite can 'shuffle' between binding positions of res during non-denaturing PAGE, without complete dissociation of the retarded complex. A mutagenesis strategy was developed for the production and isolation of mutants of Tn3 resolvase capable of directing resolution between isolated subsite I elements. Initial attempts to produce such mutants were unsuccessful. A new vector for mutagenesis and expression of resolvase was produced and the resolution substrates, used to screen for mutants, were improved.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796826  DOI: Not available
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