A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization

The lagging strand of the replication fork is initially copied as short Oka zaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-h elicase that assembles into a ring-shaped hexamer with both DNA unwinding a nd primer synthesis activities. The primase is also required for the utiliz ation of RNA primers by T7 DNA polymerase, It is not known how many subunit s of the primase-helicase hexamer participate directly in the priming of DN A synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA un winding activity. Remarkably, this monomeric primase readily primes DNA syn thesis by T7 DNA polymerase on single-stranded templates. The monomeric gen e 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synt hesis. These results show that a single subunit of the primase-helicase hex amer contains all of the residues required for primer synthesis and for uti lization of primers by T7 DNA polymerase.