Mechanochemistry of T7 DNA helicase

Jung Chi Liao, Yong Joo Jeong, Dong Eun Kim, Smita S. Patel, George Oster, Y. J. Kim

Research output: Contribution to journalArticlepeer-review

77 Scopus citations

Abstract

The bacteriophage T7 helicase is a ring-shaped hexameric motor protein that unwinds double-stranded DNA during DNA replication and recombination. To accomplish this it couples energy from the nucleotide hydrolysis cycle to translocate along one of the DNA strands. Here, we combine computational biology with new biochemical measurements to infer the following properties of the T7 helicase: (1) all hexameric subunits are catalytic; (2) the mechanical movement along the DNA strand is driven by the binding transition of nucleotide into the catalytic site; (3) hydrolysis is coordinated between adjacent subunits that bind DNA; (4) the hydrolysis step changes the affinity of a subunit for DNA allowing passage of DNA from one subunit to the next. We construct a numerical optimization scheme to analyze transient and steady-state biochemical measurements to determine the rate constants for the hydrolysis cycle and determine the flux distribution through the reaction network. We find that, under physiological and experimental conditions, there is no dominant pathway; rather there is a distribution of pathways that varies with the ambient conditions. Our analysis methods provide a systematic procedure to study kinetic pathways of multi-subunit, multi-state cooperative enzymes.

Original languageEnglish (US)
Pages (from-to)452-475
Number of pages24
JournalJournal of molecular biology
Volume350
Issue number3
DOIs
StatePublished - Jul 15 2005

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Keywords

  • ATPase
  • Helicase
  • Pre-steady state kinetics
  • Ring
  • Sequential

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