Blanc SIMI 10 - Sciences de l'information, de la matière et de l'ingénierie : Nanosciences

Energy landsacape of Homologous Recombination by manipulation of a single DNA-Rad51 complex. – DynRec

Submission summary

CONTEXT
How a nano-searcher finds its nano-target is a general problem in statistical physics. It becomes vital when the searcher is a damaged DNA fragment trying to find its counterpart on the intact homologous chromosome. If the two copies can be paired, that intact homologous sequence can be used as a template to reconstitute the damaged DNA sequence, enabling the cell to survive without genetic mutations. This process, called Homologous Recombination (HR), is essential for the maintenance of genome integrity and is promoted by the protein Rad51 in mammals. Its involvement in the BRCA1 pathway of oncogenesis makes it of considerable interest in oncology, since its failure strongly hampers DNA damage repair.



OBJECTIVE
Homologous Recombination by Rad51 involves complex protein-DNA interactions, applying forces and torques upon the DNA molecule, which deeply modify the DNA structure. Rad51 proteins wrap around the damaged DNA, unwinding it by 15° per base pair and stretching it by 50% as compared to normal B DNA, thus forming the Nucleoprotein Filament (NpF). When the latter finds the homologous strand, it encompasses it into a 3-strands complex, also unwound and stretched. It is believed that this deformation is instrumental in sequence recognition and in the strand exchange, which is ultimately necessary to rebuild two functional DNA. The detailed molecular and energetic aspects of the whole process are still poorly known. In this project, we want to develop a new generation of magnetic tweezers in order to manipulate a single DNA molecule. This system will be able, for the first time, to determine the forces AND the torques applied by Rad51 to the DNA during the HR. The DNA elongation (dl) and torsion (d??) will be measured under a given torque (T) and a controlled stretching force (F). This simultaneous measurement will allow us to determine the work (dW = F.dl + T.d? ) developed by the Rad51 protein at each step of the recombination process.
A tool able to simultaneously measure forces and torques on single molecules will also have applications in numerous other aspects of biophysics and molecular interactions.



PROBLEMS AND MOTIVATION
HR is among the most complex enzymatic reactions. It escapes the classic methods of enzymology. Several partners are involved, at least one dsDNA and one ssDNA molecules, the Rad51 protein and a given amount of ATP. In addition the DNA molecules are long helices with specific sequences, they are entangled during the homology search and Rad51 acts cooperatively to catalyze the reaction.
It has been shown over the last ten years that the single molecule approach is more appropriate to distinctly observe the kinetics and dynamics of such a complex reaction.



APPROACH
We propose to investigate the HR with three complementary approaches:
1. Single-Molecule manipulation will allow us to follow the reaction in real time in order to determine the mechano-chemical features of the HR.
2. Kinetics of homology recognition in bulk will be used extensively to determine the kinetics of DNA association and dissociation mediated by Rad51. Sequences with different homology yields will be used to determine the effect of mismatches on the HR.
3. Theory and modelling – We will develop a simplified model to evaluate the search time distribution and we will identify the parameters experimentally accessible to verify the model.

Project coordination

Giovanni CAPPELLO (INSTITUT CURIE - SECTION DE RECHERCHE)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partnership

PCC INSTITUT CURIE - SECTION DE RECHERCHE
U3B CNRS - DR BRETAGNE ET PAYS- DE-LA-LOIRE
LPTMC UNIVERSITE PARIS VI [PIERRE ET MARIE CURIE]

Help of the ANR 655,940 euros
Beginning and duration of the scientific project: - 36 Months

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