Fluorescence Methods to study DNA Translocation and Unwinding Kinetics by Nucleic Acid Motors

摘要

Translocation of nucleic acid motor proteins (translocases) along linear nucleic acids can be studied by monitoring either the time course of the arrival of the motor protein at one end of the nucleic acid or the kinetics of ATP hydrolysis by the motor protein during translocation using pre-steady state ensemble kinetic methods in a stopped-flow instrument. Similarly, the unwinding of double-stranded DNA or RNA by helicases can be studied in ensemble experiments by monitoring either the kinetics of the conversion of the double-stranded nucleic acid into its complementary single-strands by the helicase or the kinetics of ATP hydrolysis by the helicase during unwinding. Such experiments monitor translocation of the enzyme along or unwinding of a series of nucleic acids labeled at one position (usually the end) with a fluorophore or a pair of fluorophores that undergo changes in fluorescence intensity or efficiency of fluorescence resonance energy transfer (FRET). We discuss how the pre-steady state kinetic data collected in these ensemble experiments can be analyzed by simultaneous global non-linear least squares (NLLS) analysis using simple sequential “n-step” mechanisms to obtain estimates of the macroscopic rates and processivities of translocation ad/or unwinding, the rate-limiting step(s) in these mechanisms, the average “kinetic step-size”, and the stoichiometry of coupling ATP binding and hydrolysis to movement along the nucleic acid.