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Title: Biophysical studies on Tn3 resolvase
Author: Nöllmann, Marcelo
ISNI:       0000 0001 3450 2824
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2004
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Site-specific recombination is a process employed by organisms in order to perform spatially and temporally defined rearrangement of DNA molecules, such as phage integration and excision, resolution of circular multimers, inversions for expression of alternate genes, and assembly of genes during development. Tn3R is the prototype of a family of closely related mobile genetic elements referred to as the class II or Tn3 family of transposons. Tn3 contains the genes encoding a transposase, β-lactamase, and a site-specific recombinase, Tn3 resolvase (Tn3R), that is responsible for the resolution of the cointe-grate, an intermediate in the transposition reaction. Tn3R is able to resolve in vitro supercoiled plasmids containing two 114 bp res sites in direct orientation into two smaller circular plasmids, each of them with a single res site. In this thesis, the solution properties of Tn3R were studied by sedimentation equilibrium (SE) and velocity (SV) analytical ultracentrifugation and small angle neutron scattering (SANS). Tn3R was found to be in a monomer-dimer self-association equilibrium, with a dissociation constant of kD1-2= 50 μM. SV and SANS demonstrated that the low-resolution conformation of dimeric Tn3R in solution is similar to that of γδ resolvase in the co-crystal structure by Yang and Steitz (1995), but with the DNA-binding domains in a rather extended conformation. In addition, equilibrium binding of Tn3R to the individual binding sites in res (sites I, II and III) was investigated by employing fluorescence anisotropy (FA) measurements. This revealed that site IIL (site II left end) and site III have the highest affinity for Tn3R, followed by site I, and finally by site HR (site II right end). The specificity of binding of Tn3R for non-specific DNA was assayed by competition experiments, where it was shown that the affinity of binding of Tn3R to site I is 1000 times higher than to non-specific sites. A new approach, involving a combination of rigid-body and ab initio modelling was developed for the study of the solution structure of macromolecules. At first, this approach was tested by applying it to the reconstruction of the low-resolution solution conformation of a DNA Holiday junction, based on small angle x-ray scattering and sedimentation velocity data. The scattering data were analysed in two independent ways: firstly, by rigid body modelling using previously suggested models for the Holliday junction (HJ), and secondly, by ab initio reconstruction methods. Sedimentation coefficients calculated for the models generated by both methods agreed with those determined experimentally and were compatible with the results of previous studies using different techniques, but provided a more direct and accurate determination of the solution conformation of the HJ. These results confirmed that addition of Mg2+ alters the conformation of the HJ from an extended to a stacked arrangement. The solution conformation of a stable protein-DNA complex formed by a mutant of Tn3R and DNA was studied by a similar approach. Hyperactive mutants of resolvase form a complex (X-synapse) containing two site I DNA fragments and a resolvase tetramer. The low-resolution solution structure of the purified, catalytically competent X-synapse was solved from small angle neutron and x-ray scattering data, by fitting the models constructed by rigid-body transformations of a published crystallographic structure of a resolvase dimer bound to site I to the data. This analysis revealed that the two site I fragments are on the outside of a resolvase tetramer core, and provided some information on the quaternary structure of the tetramer. Finally, the rigid-body modelling method was redesigned into a general systematic approach to retrieve the conformation of a macro molecule that simultaneously agrees with a range of experimental solution properties. In this method, generalised rigid-body modelling was combined with a Monte Carlo/simulated annealing optimisation method to search over a large range of possible conformations for the structure that best fits solution experimental properties derived from small angle scattering, fluorescence resonance energy transfer, and analytical ultracentrifugation datasets. This improved methodology was evaluated by applying it to two bulged DNA fragments with very different solution conformations.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available