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DnaG is a bacterial primase which synthesizes short RNA oligonucleotides during DNA replication. These RNA oligonucleotides serve as primers for DNA synthesis by bacterial DNA polymerase Pol III. On one of the two parental strands, called the lagging strand, the primase makes a primer every few kilobases. These primers serve a substrates for synthesis of Okazaki fragments.

DnaG associates through noncovalent interactions with bacterial replicative helicase DnaB to perform its primase activity, with about three DnaG primase proteins associating with each DnaB helicase. Primases tend to initiate synthesis at specific three nucleotide sequences on single-stranded DNA (ssDNA) strand templates, and for DnaG the sequence is GTA.

Structure
The DnaG primase is a 581 residue monomeric protein with three functional domains, according to proteolysis studies. There is an N-terminal Zinc-binding domain (residues 1-110) where a zinc ion is tetrahedrally coordinated between one histidine and three cysteine residues, which plays a role in recognizing sequence specific DNA binding sites. The central domain (residues 111-433) displays RNA polymerase activities, and is the site of RNA primer synthesis. The C-terminal domain (residues 434-581) is responsible for the noncovalent binding of DnaG to the DnaB helicase protein.

Zinc-Binding Domain


The zinc-binding domain, the domain responsible for recognizing sequence specific DNA binding sites, is conserved across all viral, bacteriophage, prokaryotic and eukaryotic DNA primases. The primase zinc-binding domain is part of the subfamily of zinc-binding domains known as the zinc ribbon. Zinc ribbon domains are characterized by two β-hairpin loops which form the zinc-binding domain. Typically, zinc ribbon domains are thought to lack α-helices, distinguishing them from other zinc-binding domains. However, in 2000 DnaG’s zinc-binding domain was crystallized from Bacillus stearothermophilus revealing that the domain consisted of a five stranded antiparallel β sheet adjacent to four α helices and a 310 helix on the c-terminal end of the domain.

The zinc-binding site of B. stearothermophilus consists of three cystine residues, Cys40, Cys61, and Cys64, and one histidine residue, His43. Cys40 and His43 are located on the β-hairpin between the second and third β sheet. Cys61 is located on the fifth β sheet, and Cys64 is on the β-hairpin between the fourth and fifth β sheet. These four residues coordinate the zinc ion tetrahedrally. The zinc ion is thought to stabilize the loops between the second and third β sheet as well as the fourth and fifth β sheet. The domain is further stabilized by a number of hydrophobic interactions between the hydrophobic inner surface of the β sheet which is packed against the second and third α helices. The outer surface of the β sheet also has many conserved hydrophobic and basic residues. These residues are Lys30, Arg34, Lys46, Pro48, Lys56, Ile58, His60 and Phe62.

DNA Binding
It is thought that the function of the zinc binding domain is for sequence specific DNA recognition. DNA primases make RNA primers which are then used for DNA synthesis. The placement of the RNA primers is not random, suggesting that they are placed on specific DNA sequences. Indeed, other DNA primases have been shown to recognize triplet sequences; the specific sequence recognized by B. stearothermophilus has not yet been identified. It has been shown that if the cystine residues that coordinate the zinc ion are mutated, the DNA primase stops functioning. This indicates that the zinc-binding domain does play a role in sequence recognition. In addition, the hydrophobic surface of the β sheet, as well as the basic residues which are clustered primarily on one edge of the sheet, serve to attract single stranded DNA, further facilitating DNA binding.

Based on previous studies of DNA binding by DNA Primases, it is thought that DNA binds to the zinc-binding domain across the surface of the β sheet, with the three nucleotides binding across three strands of the β sheet. The positively charged residues in the sheet would be able to form contacts with the phosphates and the aromatic residues would form stacking interactions with the bases. This is the model of DNA binding by the ssDNA-binding domain of replication protein A (RPA). It is logical to assume that  B. stearothermophilus’  zinc-binding domain binds DNA in a similar manner, as the residues important for binding DNA in RPA occur in structurally equivalent positions in B. stearothermophilus.

RNA Polymerase Domain
As its name suggests, the RNA polymerase domain (RNAP) of DnaG is responsible for synthesizing the RNA primers on the single stranded DNA. In-vivo, DnaG is able to synthesis primer fragments of up to 60 nucleotides, but in-vivo primer fragments are limited to approximately 11 nucleotides. During the sysnthesis of the lagging strand DnaG synthesizes between 2000 and 3000 primers at a rate of one primer per-second.

RNAP domain of DnaG has three subdomains, the N-terminal domain, which has a mixed α and β fold, the central domain consisting of a 5 stranded β sheet and 6 α helices, and finally the C-terminal domain which is made up of a 3 antiparallel helical bundle. The central domain is made up in part of the toprim fold, a fold that has been observed in many metal-binding phosphotransfer proteins. The central domain and the N-terminal domain form a shallow cleft, which makes up the active site of the RNA chain elongation in DnaG. The opening of the cleft is lined by several highly conserved basic residues: Arg146, Arg221, and Lys229. These residues are part of the electrostatically positive ridge of the N-terminal subdomain. It is this ridge that interacts with the ssDNA and helps guide it into the cleft, which consists of the metal binding center of the toprim motif on the central subdomain, and the conserved primase motifs of the N-terminal domain. The metal binding site of the toprim domain is where the primer is synthesized. The RNA:DNA duplex then exits through another basic depression.

C-Terminal Domain
Unlike both the zinc-binding domains, and the RNA polymerase domains, the C-terminal domains of DNA primases are not conserved. In prokaryotic primases, the only known function of this domain is to interact with the helicase, DnaB. Thus, this domain is called the helicase binding domain (HBD). The HBD of DnaG consists of two subdomains: a helical bundle, the C1 subdomain, and a helical hairpin, the C2 subdomain. For each of the two to three DnaG molecules that bind the DnaB hexamer, the C1 subdomains of the HBDs interact with DnaB at its N-terminal domains on the inner surface of the hexamer ring, while the C2 subdomains interact with the N-terminal domains on the outer surface of the hexamer.

Three residues in B. stearothermophilus DnaB have been identified as important for formation of the DnaB, DnaG interface. Those residues include Tyr88, Ile119, and Ile125. Tyr88 is close in proximity to, but does not make contact with, the HBD of DnaG. Mutation of Tyr88 inhibits the formation of the N-terminal domain helical bundle of DnaB, interupting the contacts with the HBD of DnaG. The hexameric structure of DnaB is really a trimer of dimers. Both Ile119 and Ile125 are burried in the N-terminal domain dimer interface of DnaB and mutation of these residues inhibits formation of the hexameric structure and thus the interaction with DnaG. One other residue that has been identified as playing a crucial role in the interaction of DnaB and DnaG is Glu15. Mutation of Glu15 does not disrupt the formation of the DnaB, DnaG complex, but instead plays a role in modulating the length of primers synthesized by DnaG.