Initiation Of DNA Replication by DNA polymerases requires primers that provide a 3′-OH terminus on the template DNA. In some particular systems, long RNA transcripts, DNA fragments, or proteins covalently linked with nucleotides function as primers for DNA synthesis, but in general this function is performed by short RNA fragments newly synthesized on the template. During DNA replication, the two antiparallel DNA strands in double-stranded DNA have two different modes of synthesis, in which one strand is synthesized continuously (leading strand) and another discontinuously (lagging strand; see also Okazaki Fragments). Since the latter mechanism requires frequent priming in certain intervals on a DNA strand, an enzyme that is able to produce short RNA primers repeatedly is an essential component of most DNA replication systems. Indeed, all cells have an essential specialized enzyme, called the primase, that synthesizes short RNA primers for DNA replication. Originally, this enzyme was discovered through studies on the initiation of replication of E. coli single-stranded DNA bacteriophages. Among more than 10 replication proteins needed for the replication of these phages, the DnaG protein was identified as a factor essential for the initiation stage. Subsequent reconstitution of the reaction with purified proteins demonstrated that the role of this protein is the production of short RNA segments on a single-stranded DNA, which is subsequently used for DNA synthesis by DNA polymerase (1).
DnaG primase is inert on its own, and additional proteins cooperate in the priming process. Reconstitution experiments revealed that a mobile protein complex called the primosome is assembled and migrates along the template (2). One major member of primosome is the DnaB DNA helicase, which interacts functionally with primase and activates the enzyme. A primosome assembly site (PAS) was isolated from the E. coli fX174 phage genome as a characteristic stem-loop structure. An ATPase named PriA recognizes the PAS and forms a complex with two other primosome assembly proteins (PriB, C). Subsequently, DnaB helicase and DnaG primase are recruited to the complex. In the case of replication origins, complexes of initiator proteins also assemble the DnaB-DnaG primosome, as reported for E. coli oriC or the lambda phage origin (for a review, see (3)). The assembled primosome migrates on the template strand in a 5′ to 3′ direction, hydrolyzing ATP and synthesizing many primers. The direction of migration is opposite to that of DNA synthesis, which explains the mechanism of priming for the discontinuous, lagging strand. During DNA synthesis, the primosome will be a part of the replication fork complex and will manage the synthesis of primer RNA by the interval corresponding to the Okazaki fragment length. A similar tight link of primases and DNA helicases, and their involvement in the replication fork complexes, has also been reported in bacteriophage T4 and T7 replication (4, 5).
Eukaryotic primases have slightly different features from those of prokaryotic systems. They were detected as a component of DNA polymerase a, which is an essential DNA polymerase for chromosomal replication. This polymerase has a large subunit of about 180 kDa, a middle subunit of about 70 kDa, and two small subunits of 60 to 50 kDa (6). The primase activity is detected in the subcomplex of two small subunits, and maintenance of the complex is necessary for this activity. The primase catalytic activity is harbored in the 50-kDa subunit, and the 60-kDa one functions as its accessory subunit. Studies of the DNA elongation process using an in vitro SV40 replication reaction revealed that eukaryotes have multiple DNA polymerases and the DNA polymerase a complex is specialized for the synthesis of Okazaki fragments (7). Thus, the association of primase with DNA polymerase a would be an adapted feature of eukaryotes to synthesize Okazaki fragments efficiently by specializing one of many DNA polymerases for the priming process, although the mechanism of switching from the priming reaction to DNA synthesis in one protein complex is still unclear.
The nature of the sequence specificity of the primer RNA to initiate primer synthesis, and its ultimate length, have been studied with several E. coli replicons. The results indicate that the preferred sequence for DnaG priming is GTC, and most of the primers have pppApG at their 5′ end, which initiated from base pairing with the middle T. RNA-DNA junctions are observed at fixed sites from the primer start, and the primer length is predominantly 11 ± 1 nucleotides. Priming by T4 or T7 phage primers also has a sequence preference, but the sequences are slightly different from those of DnaG. The nature of the preferred sequence for eukaryotic primase has not been elucidated well, but the length of the primer is 12 to 14 nucleotides for Drosophila primase and 8 to 12 nucleotides for mouse or yeast primases (1).
