Primase

DNA primase is an enzyme involved in the replication of DNA and is a type of RNA polymerase. Primase catalyzes the synthesis of a short RNA (or DNA in some organisms) segment called a primer complementary to a ssDNA (single-stranded DNA) template. Primase is of key importance in DNA replication because no known replicative DNA polymerases can initiate the synthesis of a DNA strand without an initial RNA or DNA primer (for temporary DNA elongation). After this elongation, the RNA piece is removed by a 5' to 3' exonuclease and refilled with DNA. DNA primase is an enzyme involved in the replication of DNA and is a type of RNA polymerase. Primase catalyzes the synthesis of a short RNA (or DNA in some organisms) segment called a primer complementary to a ssDNA (single-stranded DNA) template. Primase is of key importance in DNA replication because no known replicative DNA polymerases can initiate the synthesis of a DNA strand without an initial RNA or DNA primer (for temporary DNA elongation). After this elongation, the RNA piece is removed by a 5' to 3' exonuclease and refilled with DNA. In bacteria, primase binds to the DNA helicase forming a complex called the primosome. Primase is activated by the helicase where it then synthesizes a short RNA primer approximately 11 ±1 nucleotides long, to which new nucleotides can be added by DNA polymerase. Archaeal and eukaryote primases are heterodimeric proteins with one large regulatory and one small catalytic subunit. The RNA segments are first synthesized by primase and then elongated by DNA polymerase. Then the DNA polymerase forms a protein complex with two primase subunits to form the alpha DNA Polymerase primase complex. Primase is one of the most error prone and slow polymerases. Primases in organisms such as E. coli synthesize around 2000 to 3000 primers at the rate of one primer per second. Primase also acts as a halting mechanism to prevent the leading strand from outpacing the lagging strand by halting the progression of the replication fork. The rate determining step in primase is when the first phosphodiester bond is formed between two molecules of RNA. The replication mechanisms differ between different bacteria and viruses where the primase covalently link to helicase in viruses such as the T7 bacteriophage. In viruses such as the herpes simplex virus (HSV-1), primase can form complexes with helicase. The primase-helicase complex is used to unwind dsDNA (double-stranded) and synthesizes the lagging strand using RNA primers The majority of primers synthesized by primase are two to three nucleotides long. There are two main types of primase: DnaG found in most bacteria, and the AEP (Archaeo-Eukaryote Primase) superfamily found in archaean and eukaryotic primases. While bacterial primases (DnaG-type) are composed of a single protein unit (a monomer) and synthesize RNA primers, AEP primases are usually composed of two different primase units (a heterodimer) and synthesize two-part primers with both RNA and DNA components. While functionally similar, the two primase superfamilies evolved independently of each other. The crystal structure of primase in E. coli with a core containing the DnaG protein was determined in the year 2000. The DnaG and primase complex is cashew shaped and contains three subdomains. The central subdomain forms a toprim fold which is made of a mixture five beta sheets and six alpha helices. The toprim fold is used for binding regulators and metals. The primase uses a phosphotransfer domain for the transfer coordination of metals, which makes it distinct from other polymerases. The side subunits contain a NH2 and COOH terminal made of alpha helixes and beta sheets. The NH2 terminal interacts with a zinc binding domain and COOH-terminal region which interacts with DnaB-ID. The Toprim fold is also found in topoisomerase and mitochrondrial Twinkle primase/helicase. Some DnaG-like (bacteria-like; InterPro: IPR020607) primases have been found in archaeal genomes. Eukaryote and archaeal primases tend to be more similar to each other, in terms of structure and mechanism, than they are to bacterial primases. The archaea-eukaryotic primase (AEP) superfamily, which most eukaryal and archaeal primase catalytic subunits belong to, has recently been redefined as a primase-polymerase family in recognition of the many other roles played by enzymes in this family. This classification also emphasizes the broad origins of AEP primases; the superfamily is now recognized as transitioning between RNA and DNA functions. Archaeal and eukaryote primases are heterodimeric proteins with one large regulatory and one small catalytic subunit. The large subunit contains a N-terminal 4Fe–4S cluster, split out in some archaea as PriX/PriCT. The large subunit is implicated in improving the activity and specificity of the small subunit. For example, removing the part corresponding to the large subunit in a fusion protein PolpTN2 results in a slower enzyme with reverse transcriptase activity.