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.
