Complex of Escherichia coli primary replicative helicase DnaB protein with a replication fork

Recognition and structure

Maria J. Jezewska, Surendran Rajendran, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous fluorescence titration, fluorescence energy transfer, and analytical ultracentrifugation methods. DnaB binds the 5' single-arm fork, the 3' single-arm fork, and the two-arm fork with stoichiometries of 1, 1, and 2 DnaB hexamers per fork, independent of the length of the duplex part of the fork. Within the structurally heterogeneous binding site, the helicase accesses most of the 20 nucleotide residues of an arm. The dsDNA of the fork does not contribute to the affinity; however, it affects the positioning of the enzyme on the 5' or 3' arm. Fluorescence energy transfer experiments provide direct evidence that the DnaB helicase binds the 5' arm of the fork in a single orientation, with respect to the duplex part of the fork. The 33- kDa domains of the hexamer face the dsDNA, while the small 12-kDa domains face the 5' end of the arm. In the complex with the 3' arm, the helicase is bound in an opposite orientation when compared to the 5' arm. This is the first determination of the strict, single orientation of a helicase in the complex with a replication fork. The 3' arm accommodates a DnaB hexamer, while another hexamer is associated with the 5' arm. The complex of two DnaB hexamers bound in opposite orientations with each arm of the fork may play an important role during bidirectional replication of the E. coli DNA.

Original languageEnglish (US)
Pages (from-to)3116-3136
Number of pages21
JournalBiochemistry
Volume37
Issue number9
DOIs
StatePublished - Mar 3 1998

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DnaB Helicases
Escherichia coli
Fluorescence
Energy Transfer
Energy transfer
Proteins
Ultracentrifugation
DNA
DNA Replication
Titration
Stoichiometry
Nucleotides
Binding Sites
Substrates
Enzymes
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Complex of Escherichia coli primary replicative helicase DnaB protein with a replication fork : Recognition and structure. / Jezewska, Maria J.; Rajendran, Surendran; Bujalowski, Wlodzimierz.

In: Biochemistry, Vol. 37, No. 9, 03.03.1998, p. 3116-3136.

Research output: Contribution to journalArticle

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