Coupling dTTP hydrolysis with DNA unwinding by the DNA helicase of bacteriophage T7

Ajit K. Satapathy, Arkadiusz W. Kulczyk, Sharmistha Ghosh, Antoine M. Van Oijen, Charles C. Richardson

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

The DNA helicase encoded by gene 4 of bacteriophage T7 assembles on single-stranded DNA as a hexamer of six identical subunits with the DNA passing through the center of the toroid. The helicase couples the hydrolysis of dTTP to unidirectional translocation on single-stranded DNA and the unwinding of duplex DNA. Phe 523, positioned in a β-hairpin loop at the subunit interface, plays a key role in coupling the hydrolysis of dTTP to DNA unwinding. Replacement of Phe 523 with alanine or valine abolishes the ability of the helicase to unwind DNA or allow T7 polymerase to mediate strand-displacement synthesis on duplex DNA. In vivo complementation studies reveal a requirement for a hydrophobic residue with long side chains at this position. In a crystal structure of T7 helicase, when a nucleotide is bound at a subunit interface, Phe 523 is buried within the interface. However, in the unbound state, it is more exposed on the outer surface of the helicase. This structural difference suggests that the β-hairpin bearing the Phe 523 may undergo a conformational change during nucleotide hydrolysis. We postulate that upon hydrolysis of dTTP, Phe 523 moves from within the subunit interface to a more exposed position where it contacts the displaced complementary strand and facilitates unwinding.

Original languageEnglish (US)
Pages (from-to)34468-34478
Number of pages11
JournalJournal of Biological Chemistry
Volume286
Issue number39
DOIs
StatePublished - Oct 30 2011
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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