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 Escherichia coli: domain alpha, amino acid residues (aa) 1-156; domain beta, aa 157-302; and domain gamma, 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 DnaBbetagamma protein, in which domain alpha was deleted, formed a hexamer; whereas the mutant DnaBalphabeta, in which domain gamma was deleted, could form only dimers. The dimerization of DnaBalphabeta was Mg(2+) 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 beta (site 1), while the other interaction site is located within domain gamma (site 2). The mutant DnaBbeta, a polypeptide of 147 aa, where both domains alpha and gamma were deleted, displayed a completely functional ATPase activity. This domain, thus, constitutes the "central catalytic domain" for ATPase activity. The ATPase activity of DnaBalphabeta was kinetically comparable to that of DnaBbeta, indicating that domain alpha had little or no influence on the ATPase activity. In both cases, the ATPase activities were DNA independent. DnaBbetagamma 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 gamma in enhancement of the ATPase activity of domain beta as well as in DNA binding. Mutant DnaBbetagamma, which lacked domain alpha, was devoid of any helicase activity pointing to a significant role for domain alpha. The major findings of this study are (i) domain beta contained a functional ATPase active site; (ii) domain gamma appeared to be the DNA binding domain and a positive regulator of the ATPase activity of domain beta; (iii) although domain alpha 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.

Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis

“…Evidently, DnaBMut2 was able to provide the functions of the helicase such as translocation on ssDNA template required for successful stimulation of the primase activity. These results were probably a consequence of the fact that mutations R324A and R326A in the RSRARR DNA binding motif of the helicase did not abolish, but significantly reduced the DNA binding and the DNA-dependent ATPase and helicase activities (46). In contrast, DnaBMut1 failed to stimulate the primase activity of the general priming system (Fig.…”

“…Our previous studies suggested that DnaBMut1, with specific mutations R328A and R329A in the RSRARR DNA binding motif exhibit a significant decrease in DNA-dependent ATPase activity and a complete loss of the helicase activity, indicating the important roles of these residues in DNA binding and helicase activity (46). Besides specific mutations, R324A and R326A of DnaBMut2 led to a considerably attenuated DNA binding as well as reduced DNA-dependent ATPase and helicase activities (46). We further addressed the question of how the mutations in RSRARR DNA binding will interfere with the primer synthesis of the general priming system.…”

“…On the other hand, the DNA binding site restricted to the motif RSRARR and the leucine zipper motif that is responsible for DnaB dimerization are localized in the opposite C-terminal domain of the DnaB helicase (46). Our previous studies suggested that DnaBMut1, with specific mutations R328A and R329A in the RSRARR DNA binding motif exhibit a significant decrease in DNA-dependent ATPase activity and a complete loss of the helicase activity, indicating the important roles of these residues in DNA binding and helicase activity (46). Besides specific mutations, R324A and R326A of DnaBMut2 led to a considerably attenuated DNA binding as well as reduced DNA-dependent ATPase and helicase activities (46).…”

“…2B, lanes 2-6). Therefore, it appeared that mutations R328A and R329A in the RSRARR DNA binding motif of DnaB not only repress the DNA binding and the helicase activity (46), but also attenuate its ability to cooperate with the primase during in vitro primer synthesis.…”

“…2A). This result is due to the fact that mutations R324A and R326B do not abolish, but rather significantly reduce, DNA binding and DNA-dependent ATPase and helicase activity (46). It has been shown that the DNA binding motif, RSRARR, is located in the C-terminal domain of E. coli helicase (45), whereas the primase binding site is positioned in the N-terminal domain (35).…”