Objective
Homologous recombination is a strand exchange between two DNA molles of identical or similar sequence. The reaction plays a crucial role in the DNA repair and DNA segregation. The reaction is catalyzed by recA protein in E. coli and its homologue, rad51 protein, in eukaryotes. Complete understanding of the reaction on structural basis requires detailed information about the structures of recA protein/DNA complexes. One of the participants previously showed that recA protein can interact with very short oligonucleotides with high affinity and catalyze the strand exchange when the oligonucleotides are tagged with a fluorescent dye (Volodin et al., FEBS Letters, 1994).
This system provides a nice experimental tool for the study of homologous recombination, which is now exploited also in biotechnology.
The fluorescence of fluorescence dye could be used to monitor the reaction
in situ.
The fluorescence dye also provides structural information of various
reaction intermediates.
Possibility to use very short oligonucleotides simplifies the reaction
analysis by separating the initiation step from the elongation step of the
reaction.
Use of short oligonucleotide could facilitate the use of various physical
methods for structural analysis. The present project aims to determine the mechanism of homologous recombination using this chemically modified oligonucleotides and also understand how the chemical modification facilitates the interaction of oligonucleotide with RecA to optimize application of the reaction in biotechnology. 1) The kinetic parameters of the reaction will be determined by fluorescence measurements of the dye-labelled oligonucleotides along with the analysis of the products by gel electrophoresis. The association rates of the first as well as the second DNA to RecA will be determined. 2) Detailed structure of the complexes will be investigated by spectroscopic methods which are previously used for the analysis of RecA-DNA complexes. Also biochemical methods such as chemical interference and crosslinking analyses of the complex will be used. This allows to determine the structure of oligonucleotide and the exact contact points between the protein and the oligonucleotides in various reaction intermediates. 3) Several modification of oligonucleotide will be made to confirm the conclusion and to find out more efficient oligonucleotides for further structural analysis and also for biotechnological application. This project includes experts of different fields: experts in RecA protein biochemical studies, spectroscopy and chemical synthesis of oligonucleotide. This multidisciplinary collaboration permits efficient advancement of the project.
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Programme(s)
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Multi-annual funding programmes that define the EU’s priorities for research and innovation.
Topic(s)
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Calls for proposals are divided into topics. A topic defines a specific subject or area for which applicants can submit proposals. The description of a topic comprises its specific scope and the expected impact of the funded project.
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Funding Scheme
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Coordinator
France
The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.