Primase

Primers are short RNA sequences that are synthesised by the enzyme primase. These primers are used to start the synthesis of DNA. Primase is classified as an RNA polymerase since it generates RNA molecules. Primase works by generating complementary short RNA sequences to a single-stranded DNA template. Primers must be created by primase before DNA replication can begin. Because DNA polymerases, the enzymes that create DNA, can only connect new nucleotides to an existing strand of nucleotides, this is the case. As a result, primase acts as a catalyst for DNA synthesis by priming and laying the groundwork.

Function of Primase

Primase binds to the DNA helicase in bacteria, generating the primosome complex. The helicase activates primase, which subsequently produces a short RNA primer of about 11 nucleotides in length, to which DNA polymerase can add new nucleotides. Primases in archaea and eukaryotes are heterodimeric proteins with a large regulatory subunit and a small catalytic subunit.

Primase synthesises the RNA segments, which are then lengthened by DNA polymerase. The alpha DNA Polymerase primase complex is formed when the DNA polymerase creates a protein complex with two primase subunits. Primase is one of the slowest and most error-prone polymerases. Primases in organisms like E. coli produce roughly 2000 to 3000 primers each second at a rate of one primer per second.

Primase also works as a pausing mechanism, stopping the replication fork from progressing and preventing the leading strand from surpassing the lagging strand. The formation of the first phosphodiester bond between two molecules of RNA is the rate-determining step in primase.

Distinct bacteria and viruses have different reproduction methods, but in viruses like the T7 bacteriophage, the primase covalently links to the helicase. Primase can form complexes with helicase in viruses like the herpes simplex virus (HSV-1). The primase-helicase complex unwinds dsDNA (double-stranded DNA) and uses RNA primers to manufacture the lagging strand. Primers generated by primase are typically two to three nucleotides long.

Structure of a primase 

Primase is an RNA polymerase that synthesizes RNA primers during DNA replication and is dependent on ssDNA. Primase, like all DNA and RNA polymerases, has structural and functional characteristics that aid in polymer elongation. It possesses structural and functional properties for commencing chain synthesis as an RNA polymerase. It has structural and functional characteristics that allow it to initiate chain synthesis on ssDNA as a primase. The structure of Escherichia coli primase, which binds zinc, at least three magnesium ions, and DnaB helicase, has been determined using amino acid sequence analysis. The active magnesium motif seen in all DNA and RNA polymerases is similar to one of the magnesium binding motifs.

This motif is thought to be important in the creation of phosphodiester bonds. The area containing the putative zinc-binding motif is the most conserved, with almost 25% of identical residues across all bacterial primases. The zinc finger’s role could be to bind ssDNA in a sequence-specific manner. Primase contains the “RNAP” motif, which is prevalent in all RNA polymerases and may play a role in chain initiation. Several in vitro test techniques have replicated many of the discoveries about primer synthesis initiation in vivo. The fact that Okazaki fragments are usually begun in vivo from d(CTG) is significant. In vitro, pure primase has been demonstrated to have this trinucleotide initiation selectivity as an inherent characteristic.

How does primase know the binding site? 

The fit between primase and the template DNA may influence primase’s recognition of the initiation site. A readout mechanism for DNA recognition has been proposed, emphasising the link between DNA sequence and shape and the enrichment of arginines in the protein’s binding site; it is thought that a set of positive charges in the protein can recognise complementary DNA shape28. The primase may similarly recognise the trinucleotide initiation site, with the binding site acting as a scanner, capturing the best template sequence to start priming. Due to structural or conformational restrictions of primase and the unique DNA sequence, the specific fit between primase and initiation site has been conserved in each bacterium.

CONCLUSION

At the replication fork, enzymes called DNA primases must be active at all times. They catalyse the creation of tiny RNA molecules that act as primers for DNA polymerases. Primers, which are formed of ribonucleoside triphosphates, range in length from four to fifteen nucleotides. The majority of DNA primases can be classified into two groups. The first category includes enzymes found in bacteria and bacteriophages that are linked to replicative DNA helicases. These bacterial primases have three unique domains: an amino-terminal domain with a zinc ribbon motif that interacts with a DNA helicase, a middle RNA polymerase domain, and a carboxyl-terminal area that is either a DNA helicase or interacts with one.