Asynchrony of Base-Pair Breaking and Nucleotide Releasing of Helicases in DNA Unwinding

Helicases harness the energy of nucleotide triphosphate hydrolysis to unwind double-stranded DNA (dsDNA) in discrete steps. In spite of intensive studies, the mechanism of stepping is still poorly understood. Here, we applied single-molecule fluorescent resonant energy transfer to characterize the stepping of two nonring helicases, Escherichia coli RecQ ( E. coli RecQ) and Saccharomyces cerevisiae Pif1 (ScPif1). Our data showed that when forked dsDNA with free overhangs are used as substrates, both E. coli RecQ and ScPif1 unwind the dsDNA in nonuniform steps that distribute over broad ranges. When tension is exerted on the overhangs, the overall profile of the step-size distribution of ScPif1 is narrowed, whereas that of E. coli RecQ remains unchanged. Moreover, the measured step sizes of the both helicases concentrate on integral multiples of a half base pair. We propose a universal stepping mechanism, in which a helicase breaks one base pair at a time and sequesters the nascent nucleotides and then releases them after a random number of base-pair breaking events. The mechanism can interpret the observed unwinding patterns quantitatively and provides a general view of the helicase activity.