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 mono- and 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.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1992|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology