Properties and Action of Tn3 Resolvase

Genetics

Brown, Janet Lesley

University:University of Glasgow

关键词: Genetics;   

Others  :  http://theses.gla.ac.uk/77366/1/10991744.pdf

来源: University of Glasgow

【 摘 要 】

Under standard in vitro reaction conditions resolvase-mediated recombination is strictly intramolecular, acting only on supercoiled substrates with 2 directly repeated res sites to generate singly linked catenated products. These unique properties are not explicable by random collision of the res sites, making the mechanism of site synapsis an intriguing problem. This study has attempted to distinguish between a number of proposed models for site synapsis. A wide range of multiple re3 site constructs were made and the resolution characteristics examined. The results cannot be accounted for by the 'tracking' model for synapsis, based on 1-dimensional diffusion of resolvase along the DNA intervening between the 2 res sites. The resolution characteristics of these multiple res site plasmids are explicable by a 'pairing model' which arises as a natural consequence of the '2-step' model for site synapsis; the bias against non-adjacent events can be explained if there is a tendency to maximise the number of synapsed sites and if there are conformational difficulties in forming more than one non-adjacent synapse. The bias can be relieved by increasing the number of intervening sites; non-adjacent events can occur at a frequency equal to adjacent events when the recombining sites are separated by 2 intervening sites on either side. Inverted sites have been shown to cause 'shadowing' in the same way as directly repeated sites. Methylation protection experiments have shown resolvase recognises and binds to each of the 6 half sites of res in essentially the same way, making major groove interactions with bases of the concensus sequence. Photofootprinting results also imply a similar mode of resolvase binding at each subsite and a similar although not identical pattern of changes in the base stacking and helix geometry at the centres of each of the subsites. It is proposed that these changes represent resolvase-induced bending of the res site DNA. These resolvase-induced changes in base photoreactivity are also detected in in vivo photofootprinting experiments.