Journal: Nucleic Acids Research

Article Title: Visualizing helicases unwinding DNA at the single molecule level

doi: 10.1093/nar/gkq173

Figure Lengend Snippet: DNA unwinding by AddA N B N is characterized by pauses and bursts of activity. ( A ) Example of how a single time-course can be segmented into pauses and unwinding phases. The raw intensity data (light blue open circle) were smoothed by running average using an optimum window size (shown as pink and dark blue lines). Then, the first derivative at every time point was calculated (grey open circle). If the derivative trace (grey open circle) is smoothed using an median filter (black line), then the histogram of the smoothed derivative values (inset) reveals two populations, one corresponding the pauses (clustered around zero) and one to the unwinding phases (clustered around 17 cpp). Thresholding ( Thr. ) of the derivative data (dashed line) allows the original intensity trace to be segmented into pauses (< Thr. , pink lines) and unwinding phases (> Thr. , dark lines). ( B–E ) To automatically analyze all data, a custom-written PERL algorithm was used (see ‘Materials and methods’ section). Using this algorithm, the distributions of the number of pauses per event (B), the duration of pauses (C), maximum rate of unwinding (D) and duration of unwinding phase (E) were obtained for 1 mM ATP (circle) and 3 μM ATP (triangle). All distributions are presented as percentage frequency, normalized to the maximum.

Article Snippet: To identify and characterize the pauses and unwinding phases a custom-written PERL algorithm (ActiveState Software Inc.) was developed.

Techniques: Activity Assay