Kinetics of ATP hydrolysis during the DNA helicase II-promoted unwinding of duplex DNA

Abstract

The ATP hydrolysis activity of DNA helicase II from Escherichia coli was examined in the presence of linear single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA). In the presence of ssDNA, the ATP hydrolysis reaction followed a linear time course until the ATP was depleted. In the presence of dsDNA, in contrast, there was a kinetic lag before a linear phase of ATP hydrolysis was achieved. The nonlinear kinetics of the dsDNA-dependent ATP hydrolysis reaction could be modeled by a kinetic scheme in which helicase II undergoes a time-dependent transition from an ATPase-inactive to an ATPase-active form. Order of addition experiments indicated that this transition was not due to a rate-limiting association event between helicase II and any other component of the reaction. Instead, agarose gel assays showed that progressive unwinding of the dsDNA occurs during the same time period as the lag phase of the ATP hydrolysis reaction. No significant ATP hydrolysis was observed when the linear dsDNA was replaced with closed circular dsDNA, suggesting that the ATP hydrolysis reaction requires a dsDNA substrate that can be unwound to the complementary single strands. These results are consistent with a model in which the lag phase of the dsDNA-dependent ATP hydrolysis reaction corresponds to progressive unwinding of the dsDNA, with the ATP hydrolysis reaction arising from helicase II molecules that are bound to the separated single strands.