Kinetic Modeling of DNA Unwinding

The DNA unwinding kinetics were fit to the n-step model (Ali and Lohman, 1997 (link)) using gfit and model [unwinding.m] in MATLAB with Optimization toolbox (The MathWorks, Inc., Natick, MA) (Levin et al., 2009 (link)). Unwinding is modeled as a multistep process with equal step-size (s) and rate constant (k_i) that are estimated from fittings as described previously (Pandey et al., 2010 (link)). More information about the fitting is provided in the Appendix—Methods section. The average unwinding rates were plotted against dNTP concentration and fit to the hyperbolic equation to obtain k_cat and K_m values. $unwinding\hspace{0.17em}rate=\hspace{0.17em}\frac{k_{cat}\ast \left[dNTP\right]}{K_m+\left[dNTP\right]}.$