Kinetics of Escherichia coli helicase II-catalyzed unwinding of fully duplex and nicked circular DNA.
Escherichia coli helicase II (UvrD) protein can initiate unwinding of duplex DNA at blunt ends or nicks, although these reactions require excess protein. We have undertaken kinetic studies of these reactions in order to probe the mechanism of initiation of unwinding. DNA unwinding was monitored directly by using agarose gel electrophoresis and indirectly through the rate of ATP hydrolysis by helicase II in the presence of an ATP-regenerating system. In the presence of fully duplex DNA and excess helicase II, the rate of ATP hydrolysis displays a distinct lag phase before the final steady-state rate of hydrolysis is reached. This reflects the fact that ATP hydrolysis under these conditions results from helicase II binding to the ssDNA products of the unwinding reaction, rather than from an intrinsic duplex DNA-dependent ATPase activity. Unwinding of short blunt-ended duplex DNA (341 and 849 base pairs) occurs in an "all-or-none" reaction, indicating that initiation of unwinding by helicase II is rate-limiting. We propose a minimal mechanism for the initiation of DNA unwinding by helicase II which includes a binding step followed by the rate-limiting formation of an initiation complex, possibly involving protein dimerization, and we have determined the phenomenological kinetic parameters describing this mechanism. Unwinding of a series of DNA substrates containing different initiation sites (e.g., blunt ends, internal nicks, and four-nucleotide 3' vs 5' ssDNA flanking regions) indicates that the rate of initiation is slowest at nicks and, surprisingly, at ends possessing a four-nucleotide 3' ssDNA flanking region.