Functional and structural heterogeneity of the DNA binding site of the Escherichia coli primary replicative helicase DnaB protein

Maria J. Jezewska, Surendran Rajendran, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

70 Citations (Scopus)

Abstract

The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative fluorescence titration, centrifugation, and fluorescence energy transfer techniques. Nuclease digestion of the enzyme- single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site. The total site is built of two subsites differing in structure and affinity, although both occlude ~10 nucleotides. ssDNA affinity for the strong subsite is ~3 orders of magnitude higher than that for the weak subsite. Flourescence energy transfer experiments provide direct proof that the DnaB hexamer binds ssDNA in a single orientation, with respect to the polarity of the sugar-phosphate backbone. This is the first evidence of directional binding to ssDNA of a hexameric helicase in solution. The strong binding subsite is close to the small 12-kDa domains of the DnaB hexamer and occludes the 5'-end of the ssDNA. The strict orientation of the helicase on ssDNA indicates that, when the enzyme approaches the replication fork, it faces double-stranded DNA with its weak subsite. The data indicate that the different binding subsites are located sequentially, with the weak binding subsite constituting the entry site for double-stranded DNA of the replication fork.

Original languageEnglish (US)
Pages (from-to)9058-9069
Number of pages12
JournalJournal of Biological Chemistry
Volume273
Issue number15
DOIs
StatePublished - Apr 10 1998

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DnaB Helicases
Single-Stranded DNA
Escherichia coli
Binding Sites
DNA
Proteins
Energy Transfer
Energy transfer
Digestion
Fluorescence
Sugar Phosphates
Centrifugation
Enzymes
DNA Replication
Titration
Nucleotides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional and structural heterogeneity of the DNA binding site of the Escherichia coli primary replicative helicase DnaB protein. / Jezewska, Maria J.; Rajendran, Surendran; Bujalowski, Wlodzimierz.

In: Journal of Biological Chemistry, Vol. 273, No. 15, 10.04.1998, p. 9058-9069.

Research output: Contribution to journalArticle

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