MUTAnTS · A microfluidic high throughput approach to helicase biophysics
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2018-02-01 → 2020-01-31
- EU contribution
- €173,076
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
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Results in brief
A microfluidic high throughput approach to helicase biophysics
DNA is the molecule used to store genetic information. In the double-helical structure of DNA the information-bearing nitrogenous bases face inwards. In order to access the information it is often necessary to disrupt the native DNA structure. A specific type of enzymes, helicases, use the energy of nucleotide hydrolysis to unwind the double helix for a range of biological functions, such as DNA repair and replication but also for chaperone functions. In some cases a single-enzyme is able to both unwind the double helix (helicase activity) and to copy it (polymerase activity). DNA replication and repair are not only essential biological functions, they are key reactions in many biotechnological and biomedical processes, that include DNA synthesis and sequencing. Improving our understanding of these functions is bound to have impact both at the fundamental level and in applications. Mutagenesis i.e. characterizing enzyme variants with specific amino-acid substitutions has long been used as a technique to probe sequence/function mapping. However helicases and polymerases are large macromolecules typically comprising several hundreds of amino-acids and a systematic studies of the relevance of each amino acid residue to the enzyme’s activity has so-far been impossible. In order to study large numbers of enzyme variants (mutants) in parallel we resort to water-in-oil emulsions. Each tiny droplet in our emulsions is used as a separate compartment (test-tube) to study a specific mutant. Our approach relies on microfluidic emulsification, which produces extremely monodisperse and reproducible emulsions. With this method we can study, in a single experiment, billions of mutants, something impossible by standard methods. Our scientific questions have focused on the helicase activity possessed by some polymerase families, and in particular on the biophysical mechanism that underlies it.
Data: CORDIS, © European Union
Project objective
We propose a novel high-throughput method to study the chemo-mechanical coupling in molecular motors using helicases, enzymes that couple ATP hydrolysis to unwinding of the double helix, as a model system. The method combines a cell-free expression system, droplet microfluidics and fluorescent assays with next generation sequencing to test large (up to millions of elements) libraries of helicase mutants for both unwinding and ATPase activity. The aim is to identify all sequence positions that, if mutated, lead to uncoupling, i.e. abolish unwinding while preserving ATPase activity. Once these positions are identified, their distribution on the enzyme sequence and structure will be analysed to obtain insight into the coupling mechanism. Notably the methodology could be applied to systems where the classical, structure-based analysis of the chemo-mechanical coupling is not possible. We will demonstrate our method on the RecQ helicase from e. coli using existing information on this system as a means of validation. Any further insight will be a demonstration of the power of our approach. When fully developed, our method will serve as a discovery tool for the interdisciplinary community working on helicases that includes medical, biological and physical sciences. It may be applied to aspects of helicase biophysics beyond the chemo-mechanical coupling, to other enzyme families and to screen for helicase inhibitors with higher throughput and lower cost when compared to robotic mictotitre-plate techniques. The proposed work will be carried out at the Griffiths Lab (ESPCI Paris) with a secondment at the Crick Institute (London). This project integrates the training in biophysics of the Experienced Researcher with the expertise of the Host Institution on high-throughput methods and the experience of the Secondment Institution on helicases. The Experienced Researcher will be trained in next generation sequencing and fluorescence-based approaches to molecular motors.
Original text from CORDIS.
Participants
- ECOLE SUPERIEURE DE PHYSIQUE ET DECHIMIE INDUSTRIELLES DE LA VILLE DEPARIS · ParisCoordinatorFrance
Links
Data: CORDIS, © European Union