DNA helicases, first discovered in 1976, are a class of enzyme that plays an integral role in DNA replication, repair, recombination, and transcription. These processes require the formation of intermediates in which duplex DNA is unwound, resulting in partially single-stranded (ss) DNA. Since helicases are important in almost all facets of nucleic acid metabolism they share some biochemical characteristics: nucleic acid binding, nucleoside triphosphate (NTP)/deoxynucleotide triphosphate (dNTP) binding and hydrolysis, and NTP/dNTP driven unwinding of duplex nucleic acids. Figure 1. gives the frame of reference in which DnaB helicase functions in E. coli DNA replication: it unwinds the double-stranded (ds) DNA in preparation for DNA Polymerase III to replicate it.1) 2)
DnaB forms a donut-like shape around the lagging strand and translocates along it as it unwinds the DNA (see Figure 2.). DNA helicases function as molecular motors using NTP hydrolysis to unwind and separate the double helix in DNA replication. They unwind dsDNA by catalytically destabilizing the hydrogen bonds between the base pairs; this is energetically driven by aforementioned NTP hydrolysis. In these unwinding reactions, an unwinding fork propagates along the DNA from the site of initiation, resulting in an ongoing unzipping of the dsDNA (see Figure 2.). This is facilitated by the binding of single-strand binding (SSB) proteins (SSB protein). Some helicases can unwind duplex DNA in vitro at rates of 500 – 1000 base pairs per second.
The specific mechanism for how most helicases unwind DNA is still unknown, though it is clear that all helicases have some similarity i.e. the mechanisms share some aspects yet differ in some finer details. However it is known that there is a variety of DNA helicases even within a single-cell organism because they are used at various stages of the nucleic acid metabolism, that is to say, there are different structural demands for the various steps. As with many enzymes specific to prokaryotes and those found in both in prokaryotes and eukaryotes, the enzyme dnaB helicase of E. coli serves as the primary model for all dnaB helicases regardless of species. At least 14 different DNA helicases have been isolated from Escherichia Coli, six from bacteriophages, 12 from viruses, 15 from yeast, eight from plants, 11 from calf thymus, and as many as 24 from human cells. DnaB of E. coli serves as the primary helicase in the DNA replication process. While dnaB is considered to be the principle replicative helicase it has been proposed that multiple helicases may play an active role in E. coli DNA replication, with one acting on the leading strand and another on the lagging strand. Like most helicases, E. coli dnaB is an oligomer – more specifically, it is comprised of six repeating subunits (see Figure 2.). The enzyme exists as a homohexamer in which six subunits form a ringlike structure, stabilized by the coordination of magnesium cations. It is believed that the oligomeric structure of helicases may provide a simple way for them to bind to multiple sites of DNA. DnaB helicase’s activities are dependent upon its ability to interact with both an ssDNA and a dsDNA that are under ATP control. 1) 2) 5) 6) 7) 8)
DnaB’s function as a helicase is driven by NTP hydrolysis. To understand how dnaB harnesses the energy stored in nucleoside triphosphates and how this is used in its helicase activity, the structural elements that are involved in NTP and DNA binding are crucial. Within dnaB the two domains of the hexamer pack with very different symmetries to form a distinct two-layered ring structure, which are involved in binding the ssDNA. Each of three bound helicase binding domains (HBDs) stabilizes the protein in a conformation that may increase its processivity. It is thought that the six subunits form a ring- or cylinder-like structure that unwinds DNA at the replication fork by translocating along and encircling the 5’ lagging strand, while the 3’ leading strand is occluded. The subunits are depicted by alternating blue and white, and pink and white, respectively. In the top surface-representation helices linking the subunits are in yellow; in the ribbon- representation Arginine fingers are displayed as red spheres. The C-terminal domain (CTD) forms a fold that contains the NTP and DNA binding sites; the DNA binding loops are seen in blue in the top structure and the six distinct NTP binding sites are in green in the bottom structure. All six nucleotide-binding sites of the DnaB hexamer are active ATPase sites. These structural features indicate the importance of NTP binding and consequently the importance of helicase activity as it requires energy form NTP hydrolysis. 2) 7) 9)
In order for the DnaB protein in E. coli to break the hydrogen bond interactions between the guanine-cytosine and adenine-thymine pairs of duplex DNA efficiently, quickly, and with a high turnover rate, the enzyme requires the energy of the phosphodiester bond released by the hydrolysis of ATP or CTP. This NTP hydrolysis step is significantly faster for the purine than for the pyrimidine triphosphate, both in the absence and in the presence of DNA as the enzyme can undergo this reaction as both a free helicase and as the enzyme-ssDNA complex. DnaB helicase has six nucleotide-binding sites, one on each subunit, which shows ground-state preference for the purine cofactors. The nucleotide binding process is biphasic i.e. three cofactor molecules bind independently in the high-affinity phase and the next three nucleotides bind in the low-affinity phase.6) 7)
The DnaB helicase binds the ssDNA with a stoichiometry of 20 ± 3 nucleotides and the equilibrium binding site is located inside the cross channel of the hexamer as seen in the Figure 4. Though each subunit can bind the ssDNA, the DnaB hexamer effectively has a single total DNA-binding site. As seen in the figure, Dnab binds DNA in a strictly single orientation with respect to the sugar-phosphate backbone. Furthermore, the strong DNA-binding subsite occludes about 10 nucleotides at the 5’ end of the bound ssDNA, while the weak DNA-binding subsite occludes about 10 nucleotides at the 3’ end of the bound DNA. This is how DnaB latches on to the ssDNA and how it can “slide” along the strand to the unwinding fork.
Without ATP hydrolysis, the DnaB helicase binds about 20 nucleotides per hexamer and the ssDNA binds predominately to a single subunit at a time. Thus, DnaB has effectively a single binding site for ssDNA. Even though each subunit can most likely bind ssDNA, more than one subunit cannot bind to it when ssDNA is already bound by another subunit. Both the gamma-phosphate and intact ribose, but not the base moiety, are important factors in inducing allosteric interactions between the nucleotide and the ssDNA binding site on DnaB. 2) 6) 7) 10)
Knowledge of nucleotide interactions, especially ATP, with DnaB is vital for understanding the various activities of the enzyme and is necessary in proposing any mechanism for DnaB’s translocation along the ssDNA or its action in unwinding dsDNA. Since DnaB uses nucleoside triphosphate energy it is also regulated through this and other related factors. DnaB binding to ssDNA is stimulated by ribonucleoside triphosphates, analogs thereof, and Mg2+, and inhibited by ADP and deoxyribonucleoside triphosphates. ATP binding is important in order for DnaB helicase to undergo conformational changes that allow it to move along the lagging strand. Bimolecular association of ATP is followed by the reversible ATP hydrolysis and subsequent conformational transition of the enzyme-product complex. The conversion of ATP to ADP has a partial equilibrium constant of about two to five, indicating that the free energy of ATP hydrolysis is released from the enzyme-product complex in the conformational transitions of the intermediates. The binding is biphasic: First, three nucleotide molecules bind independently in the high-affinity step and the next three nucleotides bind in the low-affinity step. This process in regards to the helicase and ATP (excluding the DNA strand for now) can be simplified as follows:
DnaB + ATP ⇔ DnaB • ATP ⇔ DnaB • ADP • Pi ⇔ DnaB + ADP + Pi
According to Rajendran, et al. part of the ATP binding energy originates from induced structural changes of the DnaB-ATP complex prior to ATP hydrolysis. There is the major conformation transition of the enzyme-nucleotide complex occurs in the first binding step. Their data shows that free energy is released from the enzyme-product complex in the conformational transitions following the chemical step and before the product release. 2) 6) 10) 11)
ATP holds the energy that drives the unwinding of dsDNA but other ribonucleoside triphosphates, deoxyribonucleoside triphosphates, and ADP compete for the same six binding sites as ATP. Although DnaB forms a binary complex with ssDNA this complex is not nearly as tightly bound as the ternary complex with the addition of ATP i.e. DnaB • ssDNA • ATP. This shows that the nucleotide allosterically increases the affinity of DnaB for binding ssDNA. It is clear that ATP is the dominant nucleoside because of its abundance as an energy source, however DnaB shows a preference for GTP and the capability of accepting and hydrolyzing all NTPs in its nucleotide-binding site in vitro. It is - of course - not possible to consider this preference equally in vivo. However, the following more general statement is applicable in vivo: purine nucleotides bind about four times more tightly than pyrimidine nucleotides without preference for deoxy- versus ribonucleotides.
The initial event in the schematic cycle of the mechanism is the binding of an ATP (or another ribonucleoside triphosphate) by a binding site on one of the hexameric subunits. This binding requires Mg2+ and does not seem to be cooperative. When DnaB complexes with ATP it binds tightly to DNA – shown in the second image going clockwise from the single DnaB at the top of the above figure – and provides a signal for DnaG Primase. After the ternary complex is formed the primase becomes complexed as well, leading to the actual hydrolysis step. Hydrolysis of ATP converts the DnaB • ATP complex to the DnaB • ADP complex with a low affinity for ssDNA. This result of low affinity allows the DnaB to dissociate from DNA, which in turn enables another cycle of bind and ATP hydrolysis to occur. This constitutes a distributive mechanism. 8) 10) 11)
The physiological role of the DnaB helicase is related to its interactions with ss- and dsDNA under the control of ATP binding and hydrolysis. Understanding the interactions between DnaB and nucleotide cofactors is a requirement for understanding the activities of this enzyme.
by Jess Coulter