In this project, we propose to characterize by theoretical/computational approaches the reaction of the DNA cleavage as occurring in homing endonucleases.
By combining molecular dynamics simulations with quantum mechanical calculations in a statistical mechanical coherent approaches we plan to fully characterize the factors regulating such a reaction and describe them at atomic level of details.
Therefore, the main objective of the project are:
- To achieve mechanistic insights on the process of double-strand break as occurring in homing endonucleases
- To provide a robust framework based on the combination of theoretical techniques able to help the customized
recognition DNA sequence specificity.
The endonucleases engeneering - in principle able to targeting a transgene or gene modification - is one of the emerging techniques in gene therapy area. In fact, the use of endonucleases can potentially address gene insertion, correction and inactivation, making a very effective and versatile strategy in gene therapy. Thus, from realistic and rigorous theoretical/computational models, the key-factors responsible the endonuclease activity will be identified.
The strength of the project relies on the combination of different state of the art theoretical techniques. To gain insights in such a complex mechanism, different theoretical methodologies are planned to be used in order to provide a complete picture of the DSB.
The main points addressed by the present project are the following: i) a dynamical view of the DNA-protein interactions at an atomistic level of details and ii) a complete characterization of the specific interactions at the DNA-protein interface which ultimately determines the endonuclease activity the discrimination between substrate specificity and the protein activity in statistical mechanical terms.
Such results will be obtained by the combination of advanced molecular theories, molecular dynamics simulations and quantum mechanical calculations. It is worth to mention that we also plan to perform cleavage assays on mutated variants of the homing endonuclease chosen on the base of the computational results (in collaboration with the Dr. Rafael Molina, CSIC) which can be used to validate and support the theoretical findings (such a collaboration already provided scientific outcomings, see for example Muñoz et al. 2011, Molina et al., 2016).
The project has a high relevance in both clinical and technological issues. In fact, the high degree of specificity of
these proteins makes them the perfect tools for genome customization: endonucleases binding to its specific DNA
recognition site induces a DNA double-strand break (DSB) at a unique site in the genome of a living cell. In this
respect, one of the goal of the project is to allow a speed-up of the homing endonuclease engineering by the
combination of theoretical techniques which can be directly evaluated by experiment at relatively low costs. In
summary, the project development should lead to an efficient and fast protocol able to indicate new and innovative
strategies to design homing endonuclease derivatives.
- I. G. Muñoz et al., Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus. Nucleic Acids Res. 39 (2): 729743 (2011).
- R. Molina et al., Engineering a Nickase on the Homing Endonuclease I-DmoI Scaffold J. Biol. Chem., 290 (30) (2015).
- R. Molina et al., Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics. ACS Chemical Biology, 11 (5), pp. 1401-1407 (2016).