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New methods for the structural analysis of intermediates in Tn3 site-specific recombination
Başlık:
New methods for the structural analysis of intermediates in Tn3 site-specific recombination
Yazar:
MacDonald, Alasdair Iain, author.
ISBN:
9780438060371
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (252 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Advisors: W. T. Marshall.
Özet:
A critical step in site-specific recombination is synapsis, the bringing together of the two DNA sites that are to recombine. Synapsis by Tn3 resolvase involves the interwrapping of two res sites in a topologically defined manner. The synapse is stabilised by specific protein-DNA and protein-protein interactions. The precise architecture of this large nucleoprotein complex is as yet undetermined. A modified hydroxyl radical approach has been developed to investigate the protein-DNA interactions that take place during three steps in the formation of the synaptic complex. This involved the isolation and purification of three resolvase mutants (known as M106C, T162C and D102C) which contain unique cysteine residues at specific regions of the protein. A hydroxyl radical-generating DNA cleavage reagent (EPD) was then covalently attached to the cysteine thiol of each mutant. On initiation of hydroxyl radical formation, two of the three resolvase mutants showed a set of localised DNA cleavages on individual binding sites and a complete res site. The cleavages obtained at site I corresponded to those predicted from examination of the co-crystal structure of a gammadelta resolvase dimer bound at site I. The patterns of cleavage of each binding site within a complete res site were similar to those of the individual binding sites. A slightly higher level of cleavage at the right end of site II and the left end of site III suggested that there may be some cooperativity within res that would allow these sites to bind with a higher affinity. A set of cleavages was also observed in the site I-II spacer region, giving further evidence that this region of DNA may be looped in the resolvase-res complex. Another set of experiments investigated the ability to covalently crosslink resolvase subunits to individual res binding sites. One method involved photocrosslinking by placing a photoactivatable analogue (5-iododeoxyuridine) in the DNA. The second method investigated the possibility of crosslinking the thiol of the resolvase cysteine mutant T162C to a phosphorothioate residue placed on the DNA backbone. Photocrosslinking was more efficient at obtaining a crosslinked adduct. A synapsis assay which combined photocrosslinking and EPD-resolvase induced DNA cleavage was developed, with the aim of identifying specific interactions made by individual subunits with res in the synaptic complex. A preliminary test suggested that further optimisation of the assay will be needed to obtain definite results.
Notlar:
School code: 0547
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684806.1) | 684806-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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