Objective
We have chosen to study type II restriction endonucleases and DNA methyltransferases because they provide a rich source of information about protein-DNA interactions and because they have gained an enormous importance in Molecular Biology due to their applications in recombinant DNA technologies, genome mapping, manipulation of chromosomes and health care. Recent crystallographic studies have revealed novel DNA-binding motifs and new modes of DNA recognition.
To understand the precise mechanisms underlying sequence specific DNA recognition, these studies are extended in the present proposal to the development of novel specificities and include structural and biochemical work with mutated proteins complexed with DNA, along with a systematic examination of the roles of individual residues and bases implicated in specific DNA binding. Moreover, biochemical and biophysical methods are employed to understand the thermodynamics and kinetics of DNA binding, and computational techniques are used for modelling protein-DNA interactions. It will be attempted to apply the experience gained from these experiments to the engineering of modified enzymes with altered specificities, focusing on the commercially interesting restriction endonucleases with 8-base pair recognition sequences. The project uses the EcoRV restriction endonuclease and the DNA methyltransferases EcoRV and dam as model systems with the prime objective to explore the possibility of altering their specificities, as this is considered the most informative approach for understanding interactions with DNA and fruitful in terms of potential applications. To this end, the project: a) uses crystallographic studies of the above enzymes or of their variants complexed with DNA b) employs mutagenesis and advanced selection techniques to alter the specificity of wild-type enzymes c) attempts to engineer modified enzymes with altered DNA recognition specificities, focusing on 8-base pair cutters. d) employs biocomputing approaches to develop a deeper understanding of the spatial relations in protein-DNA recognition interactions. The work will be executed in interwoven cycles of random or site-directed mutagenesis of the proteins studied, selection, gene expression, biochemical and biophysical studies over several rounds until interesting enzyme variants are obtained. Structural work on those will iteratively redefine the regions to be mutated and the cycle will be started again with novel constructions. We have assembled an international team of academic and industrial laboratories with complementary expertise in of macromolecular crystallography, mutagenesis, selection techniques, gene expression, oligonucleotide synthesis, biophysical biochemical studies and biocomputing.
The work complies with the objectives of the Biotechnology programme as new structures will be determined, our understanding of the correlation between primary and tertiary structures will be improved and it will be attempted to redesign enzymatic properties using both in-vitro selection technologies, as well as rational, computer-assisted techniques.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences genetics DNA
- natural sciences earth and related environmental sciences geology mineralogy crystallography
- natural sciences biological sciences genetics chromosomes
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
- natural sciences biological sciences molecular biology
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Coordinator
71110 Heraklion
Greece
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