Lambda Exonuclease Activity Measured Using Optical Tweezers with Active Force Clamp Control

Abstract

Exonucleases are part of many genetic recombination and repair systems. We use high-resolution optical tweezers to study bacteriophage lambda exonuclease. We trap a dumbbell construct (bead-DNA-bead) in an inverted microscope by dividing a CW laser beam into a stationary trap and a steerable trap. The inter-trap distance is actively controlled with 200 kHz update rate by acousto-optical deflectors (AOD) and field programmable gate array -card. In addition, we control the sample temperature by flowing heating fluid through aluminum jacket around the trapping objective. We follow the exonuclease activity through the length change of a 48kb dsDNA template to ssDNA-form. The dsDNA tether is attached from one strand to two streptavidin coated beads, leaving one 5’ phosphorylated end free for exonuclease to start catalytic removal of mononucleotides. At low forces the length of the ssDNA is significantly shorter than the dsDNA. The template is held in a dumbbell construct at constant force with active feedback control. This allows us to follow the enzymatic activity of the lambda exonuclease over ten micrometers while simultaneously applying a constant load between 0 and 50 pN. We apply different loads on the DNA template to measure how the template tension affects the activity of the lambda exonuclease. We also measure the activity under different ambient temperatures.