DnaG is a bacterial DNA primase and is encoded by the dnaG gene. The enzyme DnaG, and any other DNA primase, synthesizes short strands of RNA known as oligonucleotides during DNA replication. These oligonucleotides are known as primers because they act as a starting point for DNA synthesis. DnaG catalyzes the synthesis of oligonucleotides that are 10 to 60 nucleotides (the fundamental unit of DNA and RNA) long, however most of the oligonucleotides synthesized are 11 nucleotides. DnaG synthesizes a single RNA primer at the origin of replication. This primer serves to prime leading strand DNA synthesis. For the other parental strand, the lagging strand, DnaG synthesizes an RNA primer every few kilobases (kb). These primers serve as substrates for the synthesis of Okazaki fragments.
In E. coli DnaG associates through noncovalent interactions with bacterial replicative helicase DnaB to perform its primase activity, with three DnaG primase proteins associating with each DnaB helicase to form the primosome. DnaG performs this catalysis near the replication fork that is formed by DnaB helicase during DNA replication. DnaG must be complexed with DnaB in order for it to catalyze the formation of the oligonucleotide primers. Prior to the binding of any NTPs to form the RNA primer, the ssDNA template sequence binds to DnaG. The ssDNA contains a three nucleotide recognition sequence that recruits NTPs based on Watson-Crick base pairing. The two NTP binding sites on DnaG are referred to as the initiation site and elongation site. The initiation site is the site at which the NTP to be incorporated at the 5' end of the primer binds. The elongation site binds the NTP that is added to the 3' end of the primer.
Once two nucleotides are bound to the primase, DnaG catalyzes the formation of a dinucleotide by forming a phosphodiester bond via dehydration synthesis between the 3' hydroxyl of the nucleotide in the initiation site and the α-phosphate of the nucleotide in the elongation site. This reaction results in a dinucleotide and breaking of the bond between the α and β phosphorus, releasing pyrophosphate. This reaction is irreversible because the pyrophosphate that is formed is hydrolyzed into two inorganic phosphate molecules by the enzyme inorganic pyrophosphatase. This dinucleotide synthesis reaction is the same reaction as any other enzyme that catalyzes the formation of DNA or RNA (DNA Polymerase, RNA Polymerase), therefore DnaG must always synthesize oligonucleotides in the 5' to 3' direction. In E. coli, primers begin with a triphosphate adenine-guanine (pppAG) dinucleotide at the 5' end.
In order for further elongation of the dinucleotide to occur, oligonucleotide must be moved so that the 3' NTP is transferred from the elongation site to the initiation site, allowing for another NTP to bind to the elongation site and attach to the 3' hydroxyl of the oligonucleotide. Once an oligonucleotide of appropriate length has been synthesized from the elongation step of primer synthesis, DnaG transfers the newly synthesized primer to DNA polymerase III for it to synthesize the DNA leading strand or Okazaki fragments for the lagging strand.
Zinc-Binding Domain
[[File:DnaG Zinc Binding Domain.png|thumb|Left: The structure of Bacillus stearothermophilus zinc-binding domain, with the conserved hydrophobic and basic residues shown in silver.Right: Enlarged image of the zinc-binding site showing Cys40, Cys61, Cys64 and His43 coordinating a zinc ion. Diagram rendered from PDB 1D0Q.]]
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 3<sub>10</sub> helix on the c-terminal end of the domain. During the synthesis of the lagging strand DnaG synthesizes between 2000 and 3000 primers at a rate of one primer per-second. 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.
thumb|Bacillus stearothermophilus DnaG, blue, in complex with the hexameric DnaB, green. Rendered from PDB 2R6A
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. Suramin is also known to inhibit eukaryotic DNA primase by competing with GTP, so suramin is likely to inhibit DnaG via a similar mechanism.