Bacterial DnaB Helicase
Keller Bueneman '25 and Ayman Wadud '25

Contents:

I. Introduction

The bacterial protein DnaB is a highly conserved helicase enzyme that couples NTP hydrolysis with 5’ to 3’ translocation across single-stranded DNA (ssDNA). It is a necessary component of DNA replication that unwinds the double-stranded DNA ahead of the Pol III holoenzyme in the expanding replication fork. At the beginning stage of DNA replication in bacteria, the replication initiator protein DnaA opens a bubble in the dsDNA, after which the helicase loader protein DnaC loads the DnaB helicase onto each ssDNA strand. DnaC binds each subunit of DnaB in a 1:1 ratio and rearranges DnaB into an open lock washer shape that allows it to encircle the ssDNA. Following the loading phase, DnaC is displaced by the primase DnaG that binds to the N-terminal domain (NTD) of DnaB and augments its helicase activity. The DnaB C-terminal domain (CTD) associates with the tau subunit of the clamp loader complex, thereby helping to recruit the Pol III holoenzyme to the replication fork.


II. General Structure

The DnaB helicase is a homohexameric protein with a two-tiered ring structure. Each contains two globular domains, a smaller NTD and larger CTD , separated by a flexible linker domain. The linker domain is composed of the N-linker , the linker helix , and the C-linker . The NTDs form a smaller stacked on top of a larger . In terms of symmetry, the NTD ring is said to form a trimer of dimers with three fold symmetry. When bound to DNA, DnaB adopts a , right-handed staircase conformation. The unliganded protein adopts a flat, stacked ring structure.


III. DNA Binding

Given its task of traversing the entire genome, DnaB helicase naturally employs a DNA-binding mechanism that is not base or sequence specific. Rather, the CTD of DnaB is responsible for of DNA , with each subunit binding two phosphate groups of the backbone via three hydrogen bonds. This is accomplished through a loop structurally analogous to the L1 loop of RecA. The structure shown here is DnaB bound to a 13-nucleotide strand of A-form DNA. Three key residues in the CTD of each subunit make with the phosphate groups of the DNA backbone: Gly384, Glu382, Arg381. Gly384 and Glu382 recognize the phosphate groups with their respective backbone amides, and Arg381 does so with its side chain.


IV. NTPase Activity

The structure shown is DnaB bound to five molecules of , a molecule that resembles the transition state of NTP hydrolysis. The NTP binding sites are formed at the interface between the CTDs of two adjacent subunits. There are on the protein at any given moment, because the two subunits that are furthest apart do not form a proper interface. DnaB that is not bound to DNA does not form proper NTP binding site interfaces and therefore cannot bind NTP. Five GDPs are shown bound in this crystal. The base of GDP does not make any specific contacts with the protein, explaining why the enzyme can hydrolyze any NTP. There are several important components that comprise the .

Positively charged residues known as phosphate sensors simultaneously engage and neutralize the negative charges of the γ-phosphate of NTPs and the transition state of the NTP hydrolysis reaction. In DnaB, four phosphate sensor residues interact with the transition state analog: Lys216 of the Walker A motif, Gln362 from one CTD, and Lys418 and Arg420 from the adjacent CTD. It is thought that Arg420 may serve as an allosteric switch that links ATP binding to an increase in the DNA binding affinity of DnaB. are the H-bond networks that connect adjacent DNA-binding loops and link the active site to the DNA-binding loop.


V. Proposed Translocation Mechanism

Given the right-handed spiral staircase structure that DnaB adopts when bound to DNA, it is thought that it moves across the DNA via a hand-over-hand mechanism. Upon ATP hydrolysis of the subunit at the “bottom” of the staircase, the backbone interaction is broken, and the subunit translocates 12 nucleotides to the “top” of the staircase. It then binds NTP, which restores its affinity for the next pair of phosphate groups on the ssDNA. In a stepwise fashion around the hexamer, each subunit repeats this process, moving the helicase two nucleotides up for every NTP hydrolyzed.

Fig. 1. The Hand-over-Hand Mechanism of DnaB.


Video 1. The Hand-over-Hand Mechanism of DnaB.


VI. References

Bailey, S., Eliason, W. K., & Steitz, T. A. (2007a). Structure of hexameric DnaB helicase and its complex with a domain of DnaG primase. Science, 318(5849), 459–463. https://doi.org/10.1126/science.1147353

Bailey, S., Eliason, W. K., & Steitz, T. A. (2007b). The crystal structure of the Thermus aquaticus DnaB helicase monomer. Nucleic Acids Research, 35(14), 4728–4736. https://doi.org/10.1093/nar/gkm507

Itsathitphaisarn, O., Wing, R. A., Eliason, W. K., Wang, J., & Steitz, T. A. (2012). The hexameric helicase DnaB adopts a nonplanar conformation during translocation. Cell, 151(2), 267–277. https://doi.org/10.1016/j.cell.2012.09.014

Thirlway, J. (2004). DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings. Nucleic Acids Research, 32(10), 2977–2986. https://doi.org/10.1093/nar/gkh628

Wang, G., Klein, M. G., Tokonzaba, E., Zhang, Y., Holden, L. G., & Chen, X. S. (2007). The structure of a DnaB-family replicative helicase and its interactions with primase. Nature Structural & Molecular Biology, 15(1), 94–100. https://doi.org/10.1038/nsmb1356


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