Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism.

The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lack statistical power. We present a highly-generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5'-, but not 3'-ends of biotinylated DNA.

Redß177 annealase structure reveals details of oligomerization and λ Red-mediated homologous DNA recombination.

The Redß protein of the bacteriophage λ red recombination system is a model annealase which catalyzes single-strand annealing homologous DNA recombination. Here we present the structure of a helical oligomeric annealing intermediate of Redß, consisting of N-terminal residues 1-177 bound to two complementary 27mer oligonucleotides, determined via cryogenic electron microscopy (cryo-EM) to a final resolution of 3.3 Å. The structure reveals a continuous binding groove which positions and stabilizes complementary DNA strands in a planar orientation to facilitate base pairing via a network of hydrogen bonding. Definition of the inter-subunit interface provides a structural basis for the propensity of Redß to oligomerize into functionally significant long helical filaments, a trait shared by most annealases. Our cryo-EM structure and molecular dynamics simulations suggest that residues 133-138 form a flexible loop which modulates access to the binding groove. More than half a century after its discovery, this combination of structural and computational observations has allowed us to propose molecular mechanisms for the actions of the model annealase Redß, a defining member of the Redß/RecT protein family.