T7 DNA helicase: A molecular motor that processively and unidirectionally translocates along single-stranded DNA

Dong Eun Kim, Murli Narayan, Smita S. Patel

Research output: Contribution to journalArticlepeer-review

85 Scopus citations

Abstract

DNA helicases are molecular motors that use the energy from NTP hydrolysis to drive the process of duplex DNA strand separation. Here, we measure the translocation and energy coupling efficiency of a replicative DNA helicase from bacteriophage T7 that is a member of a class of helicases that assembles into ring-shaped hexamers. Presteady state kinetics of DNA-stimulated dTTP hydrolysis activity of T7 helicase were measured using a real time assay as a function of ssDNA length, which provided evidence for unidirectional translocation of T7 helicase along ssDNA. Global fitting of the kinetic data provided an average translocation rate of 132 bases per second per hexamer at 18°C. While translocating along ssDNA, T7 helicase hydrolyzes dTTP at a rate of 49 dTTP per second per hexamer, which indicates that the energy from hydrolysis of one dTTP drives unidirectional movement of T7 helicase along two to three bases of ssDNA. One of the features that distinguishes this ring helicase is its processivity, which was determined to be 0.99996, which indicated that T7 helicase travels on an average about 75 kb of ssDNA before dissociating. We propose that the ability of T7 helicase to translocate unidirectionally along ssDNA in an efficient manner plays a crucial role in DNA unwinding.

Original languageEnglish (US)
Pages (from-to)807-819
Number of pages13
JournalJournal of molecular biology
Volume321
Issue number5
DOIs
StatePublished - 2002

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Keywords

  • DNA translocation
  • Fluorescence stopped-flow
  • Hexameric helicase
  • Motor protein
  • Presteady-state kinetics

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