Pre-steady-state chemical quenched-flow techniques severe used to study DNA unwinding catalyzed by Escherichia coli UvrD helicase (helicase II), a member of the SF1 helicase suyerfamily. Single turnover experiments, with respect to unwinding of a DNA oligonucleotide, were used to examine the DNA substrate and UvrD concentration requirements for rapid DNA unwinding by pre-bound UvrD helicase. In excess UvrD at low DNA concentrations (1 nM), the bulk of the DNA is unwound rapidly by pre-bound UvrD complexes upon addition of ATP, but with time-courses that display a distinct lag phase for formation of fully unwound DNA, indicating that unwinding occurs in discrete steps, with a 'step size' of four to five base-pairs as previously reported. Optimum unwinding by pre-bound UVrD-DNA complexes requires a 3' single-stranded (ss) DNA tail of 36-40 nt, whereas productive complexes do not form readily on DNA with 3'-tail lengths, ≤ 16 nt. A 5'-ssDNA tail is neither sufficient nor does it stimulate unwinding, even in the presence of a 3'-ssDNA tail. Nitrocellulose filter binding studies show that UvrD binding affinity also increases with increasing 3'-ssDNA tail length, showing apparent positive cooperativity for binding to DNA with a 40 nt 3'-ssDNA tail. Single turn over DNA unwinding experiments performed at higher DNA concentrations (50 nM) show a sigmoidal dependence of the extent of unwinding on the pre-incubated [UvrD], also indicating cooperativity. These results indicate that the form of the UvrD helicase with optimal helicase activity is oligomeric with at least two sites for binding the DNA substrate, where one site contacts regions of the 3'-ssDNA tail that are distal from the single-stranded/double-stranded DNA junction.
- Pre-steady-state kinetics