Mechanism of DnaB Helicase of Escherichia coli: Structural Domains Involved in ATP Hydrolysis, DNA Binding, and Oligomerization

Abstract
We describe the delineation of three distinct structural domains of the DnaB helicase of Escherichiacoli: domain α, amino acid residues (aa) 1−156; domain β, aa 157−302; and domain γ, aa 303−471. Using mutants with deletion in these domains, we have examined their role(s) in hexamer formation, DNA-dependent ATPase, and DNA helicase activities. The mutant DnaBβγ protein, in which domain α was deleted, formed a hexamer; whereas the mutant DnaBαβ, in which domain γ was deleted, could form only dimers. The dimerization of DnaBαβ was Mg2+ dependent. These data suggest that the oligomerization of DnaB helicase involves at least two distinct protein−protein interaction sites; one of these sites is located primarily within domain β (site 1), while the other interaction site is located within domain γ (site 2). The mutant DnaBβ, a polypeptide of 147 aa, where both domains α and γ were deleted, displayed a completely functional ATPase activity. This domain, thus, constitutes the “central catalytic domain” for ATPase activity. The ATPase activity of DnaBαβ was kinetically comparable to that of DnaBβ, indicating that domain α had little or no influence on the ATPase activity. In both cases, the ATPase activities were DNA independent. DnaBβγ had a DNA-dependent ATPase activity that was kinetically comparable to the ATPase activity of wild-type DnaB protein (wtDnaB), indicating a specific role for C-terminal domain γ in enhancement of the ATPase activity of domain β as well as in DNA binding. Mutant DnaBβγ, which lacked domain α, was devoid of any helicase activity pointing to a significant role for domain α. The major findings of this study are (i) domain β contained a functional ATPase active site; (ii) domain γ appeared to be the DNA binding domain and a positive regulator of the ATPase activity of domain β; (iii) although domain α did not have any significant effect on the ATPase, DNA binding activities, or hexamer formation, it definitely plays a pivotal role in transducing the energy of ATP hydrolysis to DNA unwinding by the hexamer; and (iv) all three domains are required for helicase activity.