Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme

A fluorescence assay was used to measure the processivity of Escherichia coli recBCD enzyme helicase activity. Under standard conditions, recBCD enzyme unwinds an average of 30 ± 3.2 kilobase pairs (kb)/ DNA end before dissociating. The average processivity (P_obs) of DNA unwinding under these conditions is 0.99997, indicating that the probability of unwinding another base pair is 30,000-fold greater than the probability of dissociating from the double-stranded DNA. The average number of base pairs unwound per binding event (N) is sensitive to both monoand divalent salt concentration and ranges from 36 kb at 80 mM NaCl to 15 kb at 280 mM NaCl. The processivity of unwinding increases in a hyperbolic manner with increasing ATP concentration, yielding a K_N value for ATP of 41 ± 9 μM and a limiting value of 32 ± 1.8 kb/ end for the number of base pairs unwound. The importance of the processivity of recBCD enzyme helicase activity to the recBCD enzyme-dependent stimulation of recombination at Chi sites observed in vivo is discussed.