ATPase activity of Escherichia coli Rep helicase crosslinked to single-stranded DNA: implications for ATP driven helicase translocation.
To examine the coupling of ATP hydrolysis to helicase translocation along DNA, we have purified and characterized complexes of the Escherichia coli Rep protein, a dimeric DNA helicase, covalently crosslinked to a single-stranded hexadecameric oligodeoxynucleotide (S). Crosslinked Rep monomers (PS) as well as singly ligated (P2S) and doubly ligated (P2S2) Rep dimers were characterized. The equilibrium and kinetic constants for Rep dimerization as well as the steady-state ATPase activities of both PS and P2S crosslinked complexes were identical to the values determined for un-crosslinked Rep complexes formed with dT16. Therefore, ATP hydrolysis by both PS and P2S complexes are not coupled to DNA dissociation. This also rules out a strictly unidirectional sliding mechanism for ATP-driven translocation along single-stranded DNA by either PS or the P2S dimer. However, ATP hydrolysis by the doubly ligated P2S2 Rep dimer is coupled to single-stranded DNA dissociation from one subunit of the dimer, although loosely (low efficiency). These results suggest that ATP hydrolysis can drive translocation of the dimeric Rep helicase along DNA by a "rolling" mechanism where the two DNA binding sites of the dimer alternately bind and release DNA. Such a mechanism is biologically important when one subunit binds duplex DNA, followed by subsequent unwinding.
Wong
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Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Lohman
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D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D001386:Azides; D003432:Cross-Linking Reagents; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D013388:Succinimides
D001386:Azides; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D013388:Succinimides; C506744:rep protein, E coli; C062325:sulfosuccinimidyl 6-((4-azido-2-nitrophenyl)amino)hexanoate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases