Measurements of Real-Time Replication Kinetics of DNA Polymerases on ssDNA Templates Coated with Single-Stranded DNA-Binding Proteins

Optical tweezers can monitor and control the activity of individual DNA polymerase molecules in real time, providing in this way unprecedented insight into the complex dynamics and mechanochemical processes that govern their operation. Here, we describe an optical tweezers-based assay to determine at the single-molecule level the effect of single-stranded DNA-binding proteins (SSB) on the real-time replication kinetics of the human mitochondrial DNA polymerase during the synthesis of the lagging strand.