A two-site kinetic mechanism for ATP binding and hydrolysis by E. coli Rep helicase dimer bound to a single-stranded oligodeoxynucleotide

John Hsieh, Keith J.M. Moore, Timothy M. Lohman

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20 Scopus citations


Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reactions that are coupled to ATP binding and hydrolysis. We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep 'P2S' dimer (formed with the single-stranded (ss) oligodeoxynucleotide, (dT)16), in which only one subunit of a Rep homo-dimer is bound to ssDNA. Pre-steady-state quenched-flow studies under both single turnover and multiple turnover conditions as well as fluorescence stopped-flow studies were used (4°C, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol). Although steady-state studies indicate that a single ATPase site dominates the kinetics (k(cat) = 17 (± 2) s-1; K(M) = 3 μM), pre-steady-state studies provide evidence for a two-ATP site mechanism in which both sites of the dimer are catalytically active and communicate allosterically. Single turnover ATPase studies indicate that ATP hydrolysis does not require the simultaneous binding of two ATP molecules, and under these conditions release of product (ADP-P(i)) is preceded by a slow rate-limiting isomerization (~ 0.2 s-1). However, product (ADP or P(i)) release is not rate-limiting under multiple turnover conditions, indicating the involvement of a second ATP site under conditions of excess ATP. Stopped-flow fluorescence studies monitoring ATP-induced changes in Rep's tryptophan fluorescence displayed biphasic time courses. The binding of the first ATP occurs by a two-step mechanism in which binding (k+1 = 1.5 (± 0.2) x 107 M-1 s-1, k-1 = 29 (± 2) s-1) is followed by a protein conformational change (k+2 = 23 (± 3) s-1), monitored by an enhancement of Trp fluorescence. The second Trp fluorescence quenching phase is associated with binding of a second ATP. The first ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-P(i)). Binding of the second ATP then leads to the steady-state ATP cycle. As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to maintain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding. We propose that the roles of the two ATP sites may alternate upon binding DNA to the second subunit of the Rep dimer during unwinding and translocation using a subunit switching mechanism.

Original languageEnglish
Pages (from-to)255-274
Number of pages20
JournalJournal of Molecular Biology
Issue number2
StatePublished - Apr 30 1999


  • Allostery
  • Fluorescence
  • MantATP
  • Motor protein
  • Stopped-flow


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