Kinetic mechanism for the sequential binding of two single-stranded oligodeoxynucleotides to the Escherichia coli rep helicase dimer

Escherichia coli Pep helicase is a DNA motor protein that unwinds duplex DNA as a dimeric enzyme. Using fluorescence probes positioned asymmetrically within a series of single-stranded (ss) oligodeoxynucleotides, we show that ss-DNA binds with a defined polarity to Rep monomers and to individual subunits of the Rep dimer. Using fluorescence resonance energy transfer and stopped-flow techniques, we have examined the mechanism of ss- oligodeoxynucleotide binding to preformed Rep dimers in which one binding site is occupied by a single-stranded oligodeoxynucleotide, while the other site is free (P₂S dimer). We show that ss-DNA binding to the P₂S Rep dimer to form the doubly ligated P₂S₂ dimer occurs by a multistep process with the initial binding step occurring relatively rapidly with a bimolecular rate constant of k₁ = ~2 x 10⁶ M^-1 s^-1 [20 mM Tris (pH 7.5), 6 mM NaCl, 5 mM MgCl₂, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, 4 °C]. A minimal kinetic mechanism is proposed which suggests that the two strands of ss-DNA bound to the Rep homodimer are kinetically distinct even within the P₂S₂ Rep dimer, indicating that this dimer is functionally asymmetric. The implications of these results for the mechanisms of DNA unwinding and translocation by the functional Rep dimer are discussed.