Stoichiometry and DNA unwinding by the bacteriophage T4 41:59 helicase

Kevin D. Raney, Theodore E. Carver, Stephen Benkovic

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

The bacteriophage T4 41 protein is a replicative helicase that forms a hexamer in the presence of ATP and associates with the T4 59 protein. The stoichiometry of the 41:59 helicase complex and its mechanism for DNA unwinding have been investigated using steady-state and single-turnover kinetics. A partial duplex DNA fork containing two regions of single-stranded DNA (ssDNA) of 30 nucleotides each, and 30 base pairs served as the substrate. 59 was found to increase the steady-state unwinding rate of the substrate by 200-fold over the rate of 41 alone. Maximum unwinding occurred when 59 and 41 were equimolar, revealing a 1:1 stoichiometry for the complex. Varying 41 while holding 59 constant resulted in sigmoidal kinetics suggesting strong cooperativity for formation of the 41 hexamer and providing a lower limit for hexamer assembly of 65 nM. Substrates were prepared that contained a biotin-streptavidin block in either the leading or lagging strand of the duplex region of the substrate. The first order rate constant for unwinding was reduced only when the block was placed in the lagging strand of the DNA fork, indicating that the helicase interacts primarily with the lagging DNA strand.

Original languageEnglish (US)
Pages (from-to)14074-14081
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number24
DOIs
StatePublished - Jun 25 1996

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Bacteriophage T4
Bacteriophages
Stoichiometry
DNA
Substrates
Kinetics
Streptavidin
Single-Stranded DNA
Biotin
Base Pairing
Rate constants
Proteins
Nucleotides
Adenosine Triphosphate

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Raney, Kevin D. ; Carver, Theodore E. ; Benkovic, Stephen. / Stoichiometry and DNA unwinding by the bacteriophage T4 41:59 helicase. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 24. pp. 14074-14081.
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Stoichiometry and DNA unwinding by the bacteriophage T4 41:59 helicase. / Raney, Kevin D.; Carver, Theodore E.; Benkovic, Stephen.

In: Journal of Biological Chemistry, Vol. 271, No. 24, 25.06.1996, p. 14074-14081.

Research output: Contribution to journalArticle

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