Maintenance of DNA, involving replication, repair, and recombination, requires different enzymes with a range of different activities. Development of information-rich biochemical assays that report on these activities is an important step towards our understanding of the molecular mechanisms of disease pathways such as anti-microbial resistance and cancer. Traditionally, the activity of DNA-modifying enzymes is characterised through ensemble-biochemical methods, such as gel electrophoresis and fluorimetry. These methods have the drawback of averaging over large ensembles of molecules and, therefore, provide no access to information on subpopulations, molecular mechanisms and intermediate states. However, knowledge of these properties is often crucial to understanding of the molecular processes underlying DNA metabolism. Here we present a novel single-molecule assay to rapidly characterise proteins involved in DNA metabolism. Our method is simple to implement relative to existing single-molecule experiments. We observe hundreds of individual molecules at a time and implemented a highly automated data analysis pipeline to rapidly analyse large datasets.
We use wide-field total-internal-reflection-fluorescence microscopy, coupled with microfluidic flow cells, to observe conversion of single-stranded to double-stranded DNA and vice versa on the single-molecule level. The fluorescence intensities of hundreds of individual DNA molecules are simultaneously detected and computationally synchronised to create a post-synchronised average trajectory containing detailed kinetic information. Our method is highly customisable. Theoretical possibilities include but are not limited to rapid characterisation of helicases, polymerases, complete replisomes, as well as nucleases. As a proof of concept we characterised strand-displacement DNA synthesis by the bacteriophage phi29 DNA polymerase using the S.cerevisiae single-stranded binding protein RPA as a probe for single-stranded DNA. Demonstrating the richness of information accessible through this assay, we report rate constants for DNA synthesis by the phi29 DNA polymerase and binding by RPA. Furthermore we characterised the E.coli UvrD helicase. We show unwinding kinetics as well as removal of DNA-bound proteins.