Single-Molecule Measurements of the Helicase DnaB with Magnetic Tweezers

• Main Author: Ribeck, Noah
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2011
• Subjects: Biophysics; Genetics
• ISBN: 9781124657608; 1124657606

We present a multifaceted single-molecule strategy for quantifying the physical interactions between the helicase DnaB and the DNA at a replication fork. To efficiently obtain these measurements, we have developed a method for performing multiple single-molecule manipulation experiments in parallel with magnetic tweezers. We use a microscope with a wide field of view to visualize multiple DNA-tethered paramagnetic beads and track the three-dimensional position of each bead simultaneously in real time. Nearly identical manipulation by force of all visible beads is possible by applying an external magnetic field. To mitigate the increased error caused by demagnification, we have developed a strategy based on tracking multiple fixed beads. We can simultaneously manipulate and track up to 34 DNA-tethered beads at 60 Hz. We use this strategy to study the replicative helicase for Escherichia coli, DnaB, which is a hexameric, ring-shaped motor protein that encircles and translocates along ssDNA, unwinding dsDNA in advance of its motion. The microscopic mechanisms of DnaB are unknown; further, prior work has found that DnaB's activity is modified by other replication proteins, indicating some mechanistic flexibility. To investigate these issues, we quantified translocation and unwinding by DnaB in three tethered DNA geometries held under tension. Our data support the following conclusions: 1) Unwinding by DnaB is enhanced by force-induced destabilization of dsDNA; 2) The magnitude of this stimulation varies with the geometry of the tension applied to the DNA substrate, possibly due to interactions between the helicase and the occluded ssDNA strand; 3) DnaB unwinding is interrupted by pauses, which are also dependent on force and DNA geometry; and 4) DnaB moves slower when high force is applied to the helicase-bound strand, indicating that it must perform mechanical work to compact the strand against the applied force. Our results have implications for the molecular mechanisms of translocation and unwinding by DnaB and for the means of modulating DnaB activity. However, we also describe preliminary results that indicate that the activity of another hexameric helicase, gp41 from bacteriophage T4, does not exhibit any DNA geometry dependence.