Biochemical Analysis of the Site Specific Recombinases of Yeasts and of Bacteriophage P1

Ringrose, Leonie Helen (1998). Biochemical Analysis of the Site Specific Recombinases of Yeasts and of Bacteriophage P1. PhD thesis The Open University.



The site specific recombinases FLP from Saccharomyces cerevisiae and Cre from bacteriophage P1 are finding increasing use in reverse genetics. This thesis presents an investigation and comparison of the biochemical properties of site specific recombinases, with a view to optimising their use in genomic manipulation strategies.

Chapter 3 describes the characterisation of a novel site specific recombinase, the "Kw" recombinase, from the yeast Kluyveromyces waltii. The results show that Kw shares features with FLP and Cre recombinases which make it potentially useful as a tool for genomic manipulation.

In Chapter 4, kinetic properties of FLP and Cre are compared, showing that Cre has a higher affinity for the LoxP site than FLP for the FRT. The kinetics of the full excision reaction are compared for FLP and Cre. A mathematical modelling approach is used in combination with measured DNA binding rates, to determine other parameters which best describe the recombination behaviour of each recombinase. This analysis suggests that the synaptic complex is more stable for Cre than for FLP.

In Chapter 5, results show that FLP recombinase is thermolabile in an in vitro recombination assay, whereas Cre is thermostable, and that this difference also affects DNA binding. In addition, two FLP mutants with altered thermostabilities are described.

In Chapter 6, the effect of distance between FRT sites on FLP mediated excision in vitro is examined, showing that FLP is unable to excise a substrate with 74 base pairs between its target sites, and that the optimum distance, giving the fastest initial rate of recombination, is between 400 and 700 base pairs. At a given protein to substrate ratio, recombination efficiency is greatly decreased at distances of 8kb and above.

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