Soutenance de thèse : Elsa DEL VAL

Etude de variantes de recombinase en vue du développement d’une méthode d’amplification isotherme

Doctorante : Elsa DEL VAL

Laboratoire INSA : MAP

Ecole doctorale : ED341  Évolution, Ecosystèmes, Microbiologie, Modélisation

The diagnosis of infectious and parasitic diseases is a global public health issue. The World Health Organisation (WHO) defines the "ASSURED" criterion (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, with no need for Equipment and Deliverable to those who need it) for evaluating diagnostic devices and their use in resource-limited settings. In this context, the aim of this study is to develop an isothermal amplification method, central step of the genetic diagnosis, specific to bioMérieux, using two enzymes: a recombinase RecA, central actor involved in DNA repair, and a polymerase (RPA). During the first part of the study, we studied the activity of each of the two enzymes and the compatibility of the different components of the amplification mixture. The results obtained pointed the RecA protein as the limiting element of the RPA amplification reaction with, in particular, an inhibition of RecA activity by dNTPs. Thus, during the second part of the study, we studied the biological properties of four RecA from different organisms and nine Escherichia coliRecA variants in order to find variants with properties compatible with RPA amplification and to bring information about the link between RecA structure and function. We studied their recombinogenic activity, their ability to induce the SOS response, their impact on the different cellular mechanisms and biochemical properties that may be useful for the development of biotechnological applications. We showed that Dickeya dadantii RecA had an optimum strand exchange activity at 30°C and in the presence of a dNTP mixture that inhibited Escherichia coli RecA, making this protein a good candidate for RPA development. Also, the study of P. aeruginosa RecA, D. radiodurans RecA and three variants led us to raise new hypotheses on the structure- function relationship and on the monomer-monomer interactions that perturb the activity of the protein as a whole.