Kinetic Mechanism for the Sequential Binding of Two Single-Stranded Oligodeoxynucleotides to theEscherichia coliRep Helicase Dimer
- 1 January 1998
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 37 (3), 891-899
- https://doi.org/10.1021/bi9719307
Abstract
Escherichia coli Rep 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 × 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.Keywords
This publication has 9 references indexed in Scilit:
- ATP Hydrolysis Stimulates Binding and Release of Single Stranded DNA from Alternating Subunits of the DimericE. coliRep Helicase: Implications for ATP-driven Helicase TranslocationJournal of Molecular Biology, 1996
- DNA EXCISION REPAIRAnnual Review of Biochemistry, 1996
- MECHANISMS OF HELICASE-CATALYZED DNA UNWINDINGAnnual Review of Biochemistry, 1996
- DNA helicases: Enzymes with essential roles in all aspects of DNA metabolismBioEssays, 1994
- Helicase-catalyzed DNA unwinding.Journal of Biological Chemistry, 1993
- 1 Transient-State Kinetic Analysis of Enzyme Reaction PathwaysThe Enzymes, 1992
- Escherichia coli DNA helicases: mechanisms of DNA unwindingMolecular Microbiology, 1992
- DNA-induced dimerization of the Escherichia coli Rep helicaseJournal of Molecular Biology, 1991
- Large-scale purification and characterization of the Escherichia coli rep gene productJournal of Biological Chemistry, 1989