Fluorescence methods to study DNA translocation and unwinding kinetics by nucleic acid motors.

Methods in Molecular Biology
Christopher J FischerT M Lohman

Abstract

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 nonlinear least squares (NLLS) analysis using simple sequential "n-step" mechanisms to obtain estimates of the macroscopic rates and processivi...Continue Reading

Citations

Mar 1, 2013·Nucleic Acids Research·Roberto Galletto, Eric J Tomko
Jul 17, 2016·Journal of Molecular Biology·Michael J SimonTimothy M Lohman
Jul 25, 2014·Journal of Molecular Biology·Binh NguyenTimothy M Lohman
Jun 20, 2014·Nature Protocols·Ali R ÖzeşChristopher S Fraser
Sep 24, 2013·Chemical Society Reviews·Helen Hwang, Sua Myong

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