title: Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis.
abstract: Magnetic tweezers is a versatile and easy to implement single-molecule technique that has become increasingly prevalent in the study of nucleic acid based molecular motors. Here, we provide a description of the magnetic tweezers instrument and guidelines for measuring and analyzing DNA helicase activity. Along with experimental methods, we describe a robust method of single-molecule trajectory analysis based on the Student's t-test that accommodates continuous transitions in addition to the discrete transitions assumed in most widely employed analysis routines. To illustrate the single-molecule unwinding assay and the analysis routine, we provide DNA unwinding measurements of Escherichia coli RecQ helicase under a variety of conditions (Na+, ATP, temperature, and DNA substrate geometry). These examples reveal that DNA unwinding measurements under various conditions can aid in elucidating the unwinding mechanism of DNA helicase but also emphasize that environmental effects on DNA helicase activity must be considered in relation to in vivo activity and mechanism.
authors.affiliation: Laboratory of Single Molecule Biophysics, NHLBI, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: seoly@mail.nih.gov.
authors.forename: Yeonee
authors.initials: Y
authors.lastname: Seol
authors.affiliation: Laboratory of Single Molecule Biophysics, NHLBI, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: strub@mail.nih.gov.
authors.forename: Marie-Paule
authors.initials: MP
authors.lastname: Strub
authors.affiliation: Laboratory of Single Molecule Biophysics, NHLBI, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: neumankc@mail.nih.gov.
authors.forename: Keir C
authors.initials: KC
authors.lastname: Neuman
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D009690:Nucleic Acid Conformation; D052898:Optical Tweezers; D053484:RecQ Helicases / Q000235:genetics / Q000302:isolation & purification*; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D053484:RecQ Helicases
keywords: Helicase; Magnetic tweezers; Molecular motor; RecQ; Single-molecule