ENZYMATIC CHARACTERIZATION OF THE INDIVIDUAL MAMMALIAN PRIMASE SUBUNITS REVEALS A BIPHASIC MECHANISM FOR INITIATION OF DNA-REPLICATION

The enzymatic mechanism of primase was investigated using Escherichia coli and baculoviral overexpressed mouse primase subunits, p49 and p58 . Neither of the singly purified primase subunits displayed primase ac tivity alone, but the p49 subunit was able to extend a riboprimer, ind icating that this subunit contains an RNA polymerase activity. The p58 subunit cooperated with the p49 subunit in binding the initiating pur ine to form the initial dinucleotide. After initiation, the p49 subuni t alone was sufficient to extend the growing primer, but both the rate of p49 primer extension and its stability were influenced by the p58 subunit. The K(m(ATP)) in primer synthesis on poly(dT) of the p49-p58 heterodimeric primase complex was 10-fold higher than the K(m(ATP)) of the single p49 subunit in a ribo(A) primer extension assay. In additi on, labeled ATP cross-linked to both of the individually purified subu nits but with a striking difference in affinities; cross-linking was 1 1-fold more efficient to the p49 subunit. The interaction of the two p rimase subunits with polymerase a was also investigated. Immunoprecipi tation experiments indicate that only the p58 subunit directly contact s the p180 subunit of DNA polymerase alpha. Competition experiments in the coupled primase-polymerase assay with a catalytically inactive mu tant of DNA polymerase alpha and the Klenow fragment suggest that the DNA polymerase alpha-primase complex does not dissociate from the prim er during the transition from RNA to DNA synthesis.