Direct Single Molecule Measurement of ATP Hydrolysis Substates in Hel308 DNA Helicase using Nanopore Tweezers

Single-molecule picometer resolution nanopore tweezers (SPRNT) is a single molecule tool for studying enzymes that move on nucleic acids (NA). SPRNT measures NA position relative to the enzyme at sub-Angstrom spatial resolution on millisecond timescales, while simultaneously providing the NA sequence near the enzyme. We use this method to resolve two substates of the ATP hydrolysis cycle of a Hel308 DNA Helicase. By examining the dwell times of each state, we derive a kinetic model of Hel308's translocation along single-stranded DNA, and find that the model's rate constants depend on the DNA sequence within the enzyme. This is, to our knowledge, the first evidence of sequence-dependent translocase behavior in a helicase system.