DNA replication is the process by which an organism’s DNA is used as a template for the production of another DNA strand. The function of a primase in this process is to catalyze the synthesis of a short RNA or DNA segment called a primer that is complimentary to a single stranded DNA (ssDNA) template (link to ssDNA). The importance of primase activity is owed to the lack of another initiate for the synthesis of DNA. Polymerase itself has no known initiating capability without a primer. The primer itself serves as a target for polymerase to “sit” on DNA and proceed with replication. Therefore, without a primer, there is no replication, and without a primase, there is no primer. The replicative helicase and primase in this case, DnaB and DnaG respectively, come from the bacteria Escherichia Coli.
The above image represents the theoretical model of DNA replication. It includes the initial helicase unwinding of DNA, the interaction between DnaB and DnaG, and shows how DNA is split during replication. This model of DnaB complexed with DnaG and replication fork DNA is the consensus across the field. The proteins are shown in a surface representation. DnaB consists of the (red) (DnaB C-terminal dDomain) and the (light blue) (DnaB N-terminal domain) domains, formingwhich form a trimer of dimers. DnaG is representated by the green (DnaG C-terminal HBD), pink (DnaG Polymerase domain) and yellow (DnaG N-terminal zinc binding domain).[F] The primer is shown in dark blue and the modeled DNA being replicated is shown in beige colored spheres.
This primase is composed of three main domains: a C-terminal domain (residues 434-581)[F] that binds to DnaB helicase, an N-terminal zinc-binding domain which interacts with ssDNA, and a central domain which is primarily responsible for primer synthesis.[F] There has been no 3D structure determined for full-length DNAG primase, only multiple crystal structures of the C-terminal domains among different species. There has been no research in the mapping of the N-terminal domain or the central domain.
The C-terminal domain (DnaG-C) is the only domain of E.Coli DNAG that has been mapped. Crystal structure has shown that DnaG-C is an elongated molecule composed of two globular subdomains connected by a long helix. It exists as an asymmetric dimer of subunits with different conformations, connected at different locations on the helix. These multiple binding locations allow for a certain flexibility of the helix.[E] This domain’s ternary structure consists of a helical bundle (the CI domain) terminated by a helical hairpin (the C2 subdomain).[F,G,H] The structure of DnaG-C comprises six helices arranged similar to that of as in the DnaB N-terminal domain of DnaB. The crystal structure of dimeric DnaG-C shows the two domains having different orientations and boundaries of helix 6 (Figure 2. D & E), illustrating that this helix can be separated from the core of the structure. [F] The structure of DnaG-C as a crystal structure of shows DnaG-C as a domain swapped dimer.[G] This means the helix 6 from one protein molecule binds to the core of the other(supported online reading). The average superposition of the backbone atoms of residues 447-581 of the 20 different NMR conformers of DnaG-C are shown in Figure 3. This figure also maps the observed side chain sequence of amino acids.
The polypeptide backbone is drawn as a ribbon (Figure 3.) and the flexible N-terminal 15 residues are omitted for clarity. The image also shows the amino acid side chains on DnaG-C. In Figure 2, the short 310 helix between helices 2 and 3 was found in fewer than half of the NMR conformers and so was not labeled. [A] shows E.Coli DnaG-C. [B] N-terminal domain of E. Coli DnaB (Residues 24-136). [C] DnaG-C fragment [D] Conformer I of the crystal structure dimer of E. Coli DnaG-C. E. Conformer II of the crystal structure dimer of E. Coli DnaG-C. [G]
There has been much debate between two proposed hypotheseis for the interaction of the DNAG with DNAB. The first claims that DNAB interacts with the single-stranded DNA, inducing a change in conformation to allow DNAG to load the DNA for polymerase activity. It was observed that DNA binding induced secondary structure change in ssDNA[I]. The second hypothesis is that DNAB interacts directly with DNAG and ssDNA. This would facilitate loading of the primase on the template DNA and/or activation of the enzyme[J].
The helicase-primase interaction plays a critical role in the event of DNA replication. It is mediated by the helicase interaction domain that lies within the primase.[M] This interaction coordinates the initiation of bidirectional replication at the chromosomal origin. It provides regulation of the synthesis of Okazaki fragments during lagging-strand synthesis.[A] It is believed that three positive, conserved electrostatic patches on the N-terminal of DnaB serve as potential binding sites for DNA which would guide the DNA toward the DnaG active site. [F] It is understood that the stoichiometric ratio of DnaG interacting with DnaB is 3:1. [B,C,D]
DnaB exists as a trimer of dimers stabilized by hydrophobic interactions. [F] The T7 gp4 protein of phage T7 is referred to as an analog for DnaB C-Terminal Domain (CTD). T7 gp4 contains two domains, one binding for helicase activity and the other for primase activity. Both binding domains are critical for the ability of the T7 phage to replicate.[N] Gp4 lacks a domain equivalent to the DnaB NTD [O,P] but instead has two domains related to the ZPD and RPD of DnaG attached to its N-terminus.
Comparison of the structures of DnaB and the gp4 Helicase shows that oligomerization of the proteins is very similar. Gp4 takes different orientations but provides insight into the behavior of DnaB during interaction with DnaG. Due to DnaB CTD’s orientations, most of the NTP binding sites are not near an arginine finger. [F] The arginine finger stimulates NTP hydrolysis of DnaB and orients the CTDs of DnaB to prepare its N-terminal domains for DnaG binding and replication.
For the specific helicase DnaB in E. Coli, there is strong physical and chemical interaction with the primase DnaG. The attraction of DnaB to DnaG actually stimulates both of their activities. DnaG increases both the nucleoside triphosphatase (NTPase) and helicase activities (the unwinding of the DNA) of DnaB.[S] DnaB on the other hand both increases and modulates the synthesis of the primers of DnaG.[Q] The ternary structure of the aforementioned HBD of DnaG, including the C1 subdomain helical bundle and the C2 subdomain helical hairpin, is sufficient to both bind to and stimulate the activities of DnaB.[S,F] The tertiary structure of the HBD is very similar to that of NTD folding of DnaB. [O,R]
The above image is a more detailed look at the orientation of the different domains and subdomains of DnaG in complex with DnaB. [A] is a “Top” view of a ribbon representation of the complex showing the three HBD (Helicase Binding Domain, green) bound at the periphery of DnaB’s N-terminal Domain (blue). [B] shows a “Side’’ view of a surface representation of the complex revealing no interaction between the HBD and the DnaB C-terminal domain (red). [C] illustrates the backbone trace of the HBD DnaB surface interaction, residues known to modulate the interaction between the two are shown as colored spheres. [F]
One particular research group provided evidence for this functional relationship through mutating DNAB and DNAG to reduce physical interactions that resulted in a reduction in primer synthesis. A 4- to 5- fold decrease in promoting the primer synthesis (in vitro) with mutated DNAB as opposed to wild type (wt) DnaB[H]. Other experiments have shown that the C-terminal peptide, and not the N-terminal, of DNAG interacted with DnaB[K,L]. Much of what we know about the interaction of DnaB and DnaG is still theoretical.
A. Tougu, K., and Marians, K. J. (1996) J. Biol. Chem. 271, 21398–21405
B. Mitkova AV, Khopde SM & Biswas SB (2003) Mechan- ism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis. J Biol Chem 278, 52253–52261.
C. Thirlway J, Turner IJ, Gibson CT, Gardiner L, Brady K, Allen S, Roberts CJ & Soultanas P (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 Res 32, 2977–2986.
D. Oakley AJ, Loscha KV, Schaeffer PM, Liepinsh E, Pintacuda G, Wilce MCJ, Otting G & Dixon NE (2005) Crystal and solution structures of the helicase-binding domain of Escherichia coli primase. J Biol Chem 280, 11495–11504.
E. Oakley AJ, Loscha KV, Schaeffer PM, Liepinsh E, Pintacuda G, Wilce MC, Otting G, Dixon NE. Crystal and solution structures of the helicase-binding domain of Escherichia coli primase. J. Biol. Chem. 2005;280:11495-11504
F. Bailey S, Eliason WK, Steitz TA. Structure of hexameric DnaB helicase and its complex with a domain of DnaG primase. Science 2007;318:459-463
G. Xun-Cheng Su, Patrick M. Schaeffer, Karin V. Loscha, Pamela H. P. Gan, Nicholas E. Dixon, Gottfried Otting. Monomeric solution structure of the helicase-binding domain of Ezcherichia coli DnaG primase. The FEBS Journal 2006;273;4997-5009.
H. Lu Y., Ratnakar P. V. A. L., Mohanty B. K., Bastia D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93:12902–12907
I. Kelly T J(1988) J Biol Chem 263:17889–17892.
J. Arai K-I, Kornberg A(1979) Proc Natl Acad Sci USA 76:4308–4313.
K. Tougu K, Peng H, Marians K J(1994) J Biol Chem 269:4675–4682.
L. Tougu K, Marians K J(1996) J Biol Chem 271:21391–21397.
M. Karl Syson, Jenny Thirlway, Andrea M. Hounslow, Panos Soultanas, and Jonathan P. Waltho (2005) Solution Structure of the Helicase-Interaction Domain of the primase DnaG: A Model for Helicase Activation.
N. E. A. Toth, Y. Li, M. R. Sawaya, Y. Cheng, T. Ellenberger, Mol. Cell 12, 1113 (2003)
O. S. Bailey, W. K. Eliason, T. A. Steitz, Nucleic Acids Res. 35, 4728 (2007)
P. E. A. Toth, Y. Li, M. R. Sawaya, Y. Cheng, T. Ellenberger, Mol. Cell 12, 1113 (2003)
Q. J. E. Corn, J. M. Berger, Nucleic Acids Res. 34, 4082 (2006)
R. A. J. Oakley et al., J. Biol. Chem. 280, 11495 (2005)
S. L. E. Bird, H. Pan, P. Soultanas, D. B. Wigley, Biochemistry 39, 171 (2000)