Coupling of DNA unwinding to nucleotide hydrolysis in a ring-shaped helicase

Ilker Donmez, Smita S. Patel

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

Abstract

The ring-shaped T7 helicase uses the energy of dTTP hydrolysis to perform the mechanical work of translocation and base pair (bp) separation. We have shown that the unwinding rate of T7 helicase decreases with increasing DNA stability. Here, we show that the dTTPase rate also decreases with increasing DNA stability, which indicates close linkage between chemical transition steps and translocation steps of unwinding. We find that the force-producing step during unwinding is not associated with dTTP binding, but dTTP hydrolysis or Pi release. We determine that T7 helicase extracts ∼3.7 kcal/mol energy from dTTPase to carry out the work of strand separation. Using this energy, T7 helicase unwinds ∼4 bp of AT-rich DNA or 1-2 bp of GC-rich DNA. T7 helicase therefore adjusts both its speed and coupling ratio (bp/dTTP) to match the work of DNA unwinding. We discuss the mechanistic implications of the variable bp/dTTP that indicates T7 helicase either undergoes backward movements/futile hydrolysis or unwinds DNA with a variable bp-step size; 'long and fast' steps on AT-rich and 'short and slow' steps on GC-rich DNA.

Original languageEnglish (US)
Pages (from-to)1718-1726
Number of pages9
JournalEMBO Journal
Volume27
Issue number12
DOIs
StatePublished - Jun 18 2008

ASJC Scopus subject areas

  • General Neuroscience
  • Molecular Biology
  • General Biochemistry, Genetics and Molecular Biology
  • General Immunology and Microbiology

Keywords

  • Coupling ratio
  • Helicase
  • Motor
  • Stepsize

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