Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: The effect of the 3′ arm and the stability of the dsDNA on the unwinding activity of the Escherichia coli DnaB helicase

Roberto Galletto, Maria J. Jezewska, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

48 Scopus citations

Abstract

The effect of two structural elements of a replication DNA fork substrate, the length of the 3′ arm of the fork and the stability of the double-stranded DNA (dsDNA) part, on the kinetics of the dsDNA unwinding by the Escherichia coli hexameric helicase DnaB protein has been examined under single turnover conditions using the rapid quench-flow technique. The length of the 3′ arm of the replication fork, i.e. the number of nucleotides in the arm, is a major structural factor that controls the unwinding rate and processivity of the helicase. The data show the existence of an optimal length of the 3′ arm where there is the highest unwinding rate and processivity, indicating that during the unwinding process, the helicase transiently interacts with the 3′ arm at a specific distance on the arm with respect to the duplex part of the DNA. Moreover, the area on the enzyme that engages in interactions has also a discrete size. For DNA substrates with the 3′ arm containing 14, or less, nucleotide residues, the DnaB helicase becomes a completely distributive enzyme. However, the 3′ arm is not a "specific activating cofactor" in the unwinding reaction. Rather, the 3′ arm plays a role as a mechanical fulcrum for the enzyme, necessary to provide support for the advancing large helicase molecule on the opposite strand of the DNA. Binding of ATP is necessary to engage the 3′ arm with the DnaB helicase, but it does not change the initial distribution of complexes of the enzyme with the DNA fork substrate. Stability of the dsDNA has a significant effect on the unwinding rate and processivity. The unwinding rate constant is a decreasing linear function of the fractional content of GC base-pairs in the dsDNA, indicating that the activation of the unwinding step is proportional to the stability of the nucleic acid.

Original languageEnglish (US)
Pages (from-to)101-114
Number of pages14
JournalJournal of Molecular Biology
Volume343
Issue number1
DOIs
StatePublished - Oct 8 2004
Externally publishedYes

Keywords

  • E. coli DnaB helicase
  • helicase mechanism
  • processivity
  • rapid quench-flow
  • single turnover kinetics

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Fingerprint Dive into the research topics of 'Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: The effect of the 3′ arm and the stability of the dsDNA on the unwinding activity of the Escherichia coli DnaB helicase'. Together they form a unique fingerprint.

  • Cite this