Kinetic studies on the mechanisms of DNA binding and DNA unwinding by the Escherichia coli Rep helicase

Kinetic studies were undertaken to elucidate the mechanism by which the Escherichia coli Rep helicase binds ss-DNA, dimerizes, and unwinds ds-DNA. A fluorescence assay was developed that can be used to monitor helicase-catalyzed unwinding of duplex DNA continuously in real time. Single turnover kinetic studies performed as a function of excess Rep concentration show that Rep-catalyzed unwinding of an 18 base pair duplex containing a 3{dollar}spprime{dollar}-ss-(dT){dollar}sb{lcub}20{rcub}{dollar} tail is biphasic. The observed rate constant of the first phase is independent of (Rep) and measures the rapid single turnover unwinding of the duplex DNA by Rep dimers bound in productive complexes (23 {dollar}pm{dollar} 3 base pairs s{dollar}sp{lcub}-1{rcub}{dollar} at 25.0{dollar}spcirc{dollar}C). The observed rate constant for the second phase increases linearly with (Rep), reflecting DNA unwinding that is limited by a Rep binding event occurring with a bimolecular rate constant of 1.8 {dollar}pm{dollar} 0.1 {dollar}times{dollar} 10{dollar}sp5{dollar} M{dollar}sp{lcub}-1{rcub}{dollar} s{dollar}sp{lcub}-1{rcub}{dollar} which is consistent with dimerization limiting the rate of unwinding and the dimer being the active form of the Rep helicase. The monomeric E. coli Rep protein x undergoes a DNA-induced dimerization upon binding either single stranded or duplex DNA. As a model system for the assembly of productive unwinding complexes, the minimal kinetic mechanism was determined for single stranded DNA (S) binding to Rep monomer (P) and the induced dimerization reaction using stopped-flow fluorescence. DNA binding occurs by a two-step mechanism where the bimolecular step (3.3 {dollar}pm{dollar} 0.5 {dollar}times{dollar} 10{dollar}sp7{dollar} M{dollar}sp{lcub}-1{rcub}{dollar} s{dollar}sp{lcub}-1{rcub}{dollar}) is followed by an isomerization to form PS*, which can then dimerize with P (4.5 {dollar}pm{dollar} 0.3 {dollar}times{dollar} 10{dollar}sp5{dollar} M{dollar}sp{lcub}-1{rcub}{dollar} s{dollar}sp{lcub}-1{rcub}{dollar}) to form the P{dollar}sb2{dollar}S Rep dimer. This mechanism provides direct evidence that Rep monomers can bind ss-DNA and that ss-DNA binding induces a conformational change in the Rep monomer that is probably required for Rep dimerization. The dissociation rate of a fluorescent ss-oligodeoxynucleotide bound to one subunit of the dimeric Rep helicase is stimulated by ss-DNA binding to the other subunit, and that the rate of this ss-DNA exchange reaction is further stimulated {dollar}sim{dollar}60-fold upon ATP hydrolysis. These results suggest a mechanism for ATP-driven translocation of the dimeric Rep helicase along ss-DNA.