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High-resolution tweezers for DNA replication and sequence identification

Objective

We propose to investigate the enzymes responsible for DNA replication and repair in micromanipulation experiments with a resolution of a single base. The detailed mechanism by which DNA is synthesized base after base and the coordination of the enzymes involved in this process are not fully understood. We shall develop new magnetic tweezers using lithographic techniques associated with evanescent field detection to address these issues. We shall build arrays of these devices working in parallel, each one on a single DNA molecule and where the measurement of its extension reveals enzymatic activity. The DNA molecule in these devices will form a hairpin the opening of which can be detected with a single base resolution.

We will study the different enzymes involved in DNA replication. Firstly, we wish to follow in real time the incorporation of bases one by one by a DNA-polymerase and investigate the proof-reading mechanism of this enzyme. We shall also investigate the translocation mechanisms of different helicases involved in DNA replication and repair. Finally, we plan to study the cooperative action between different enzymes involved in the replication machinery with the help of parallelized micro-tweezers: the coupling between helicase and primase in the lagging strand synthesis, the coupling between the helicase and polymerase during leading strand synthesis and the coordination between leading and lagging strand synthesis.

Moreover observing a DNA-polymerase at the single base level is the first step of a DNA sequencing method. Preliminary experiments demonstrate that the unzipping assay is a new way to determine the position of a small DNA sequence with single base resolution. We shall investigate different experimental schemes to achieve this goal.

Call for proposal

ERC-2010-AdG_20100224
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Host institution

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Address
Rue Michel Ange 3
75794 Paris
France
Activity type
Other
EU contribution
€ 1 559 647,20
Principal investigator
Vincent,jean,marie,christian Croquette (Dr.)
Administrative Contact
Julie Zittel (Ms.)

Beneficiaries (3)

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
France
EU contribution
€ 1 559 647,20
Address
Rue Michel Ange 3
75794 Paris
Activity type
Other
Principal investigator
Vincent,jean,marie,christian Croquette (Dr.)
Administrative Contact
Julie Zittel (Ms.)
UNIVERSITAT DE BARCELONA
Spain
EU contribution
€ 466 240,80
Address
Gran Via De Les Corts Catalanes 585
08007 Barcelona
Activity type
Higher or Secondary Education Establishments
Administrative Contact
Xavier Gutierrez (Mr.)
The Pennsylvania State University
United States
EU contribution
€ 167 678
Address
Technology Center Bldg. 110
16802 University Park
Activity type
Higher or Secondary Education Establishments
Administrative Contact
Lynn P. Mitchell (Mr.)