Making primers for DNA synthesis
The enzyme primase catalyzes the synthesis of the primers from which DNA polymerases can begin synthesis (Figure 5.21). Primers are short oligonucleotides, ranging from 6 to 60 nucleotides long. They can be made of ribonucleotides or a mixture of deoxyribonucleotides and ribonucleotides. The principal primase in E. coli is the 60 kDa protein called DnaG protein, the product of the dnaG gene. The major primase in eukaryotic cells is DNA polymerase \(\alpha\).
The primase of E. coli, DnaG protein, cannot synthesize primers by itself, but rather it is part of much larger complex called the primosome. The primosome acts repeatedly during lagging strand synthesis, finding a primer-binding site on the SSB-coated single-stranded template strand and synthesizing a primer. Identification of the components of the primosome was aided by the convenient model system of in vitrosynthesis of fX174 DNA. fX174 is a single-stranded bacteriophage; the DNA found in the virus is termed the plus strand. After infection of E. coli, this plus strand is converted to a double-stranded replicative form (Figure 5.24 A). The conversion of single‑stranded phage DNA to duplex DNA occurs by the synthesis of several Okazaki fragments, and hence it is a good model for discontinuous synthesis on the lagging strand. This reaction can be carried out in vitro, which allowed the biochemical dissection of the various steps in primosome assembly and movement.
|protein||gene||activities and functions|
|PriA||priA||helicase, 3' to 5' movement, site recognition|
|DnaT||dnaT||needed to add DnaB‑DnaC complex to preprimosome|
|DnaC||dnaC||forms complex with DnaB|
|DnaB||dnaB||helicase, 5' to 3' movement. DNA dependent ATPase.|
Five different proteins are found in a prepriming complex, PriA, PriB, PriC, DnaT, and DnaB (Table 5.4). A sixth protein, DnaC, is needed for the assembly of this complex. In the case of fX174 viral DNA template coated with SSB, PriA (Figure 5.24 B) recognizes a primer assembly site. The proteins PriBand PriCare then added to form a complex. The hexameric protein DnaBis in a complex with six molecules of DnaCwhen it is not on the DNA. In an ATP-dependent process, and with help from DnaT, DnaB is transferred to the template and DnaC is released.
The prepriming complex is now ready for the primase, DnaG, to bind and make the active primosome. Although the role of each of the proteins in the primosome is not yet clear, information is available on some of the steps in primosome action. The preferred binding site on the template for primase is CTG, with the T being used as the template for the first nucleotide of the primers. A high affinity complex between DnaB and ATP forms or stabilizes a secondary structure in the single-stranded template DNA that is used by primase; this is thought to be how DnaB "activates" the primase to begin synthesis. After ATP hydrolysis by DnaB, the low affinity ADP-DnaB complex dissociates from the template. The primosome can now move to the next site for primer synthesis.
Figure 5.24. Assembly and migration of the primosome. After assembly of the prepriming complex, DnaG joins the complex to complete the primosome. After synthesis of the primer (dark black line), the primosome moves to the next site for synthesis. This tracking along the SSB-coated single stranded DNA requires ATP hydrolysis and causes dissociation of some of the SSB. In the diagram, the primosome is shown moving in a 5' to 3' direction along the template strand (clockwise), which is the opposite of the direction of primer synthesis. This would be the direction of movement catalyzed by DnaB. The primosome also contains PriA, which catalyzes movement along single-stranded DNA in the opposite direction. Once primers have been synthesized, DNA polymerase III can synthesize Okazaki fragments from them, the primers are excised and gaps repaired by DNA polymerase I, and then the fragments are ligated together.
The primosome contains two helicases than can move along single-stranded DNA with opposite polarity. PriA moves in a 3' to 5' direction, whereas DnaB moves in a 5' to 3' direction. When tested in vitrowith a substrate similar to that shown in Figure 5.22, fragments from each end are displaced, indicating that the primosome moved in one direction on some molecules and in the other direction on others. Figure 5.24 B shows the migration as driven by the DnaB helicase, but movement can also occur in the other direction as well.