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J Biol Chem, Vol. 273, Issue 34, 21608-21615, August 21, 1998

Primase Activity of Human DNA Polymerase -Primase
DIVALENT CATIONS STABILIZE THE ENZYME ACTIVITY OF THE p48 SUBUNIT

Annerose Schneider, Richard W. P. Smith, Armin R. Kautz, Klaus Weisshart^§, Frank Grosse, and Heinz-Peter Nasheuer

From the Institut für Molekulare Biotechnologie,  Abteilung Biochemie und ^§ Abteilung Molekulare Cytologie und Elektronenmikroskopie, Beutenbergstrasse 11, D-07745 Jena, Germany

DNA polymerase -primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions. To study the primase activity of the complex, we expressed cDNAs encoding for the human p58 and p48 subunits either as single proteins or together using Escherichia coli expression vectors. Co-expression of both primase subunits allowed the purification of a heterodimer in high yields that revealed stable primase activity. Purified recombinant p48 subunit showed enzyme activity, whereas purified p58 did not. In contrast to the heterodimer, the primase activity of p48 was unstable. The activity of p48 could be stabilized by the addition of the divalent cations Mg²⁺ and Mn²⁺ but not Zn²⁺. On a poly(dC) template the primase activity was hardly influenced by the monovalent cation potassium. However, by using poly(dT) as a template the recombinant p48 activity was sensitive to salt, whereas recombinant p58-p48 and the bovine DNA polymerase -primase purified from thymus were less sensitive to the addition of monovalent cations. A complex of bacterially expressed primase and baculovirus-expressed p180 and p68 was assembled in vitro and shown to support replication of simian virus 40 DNA in a cell-free system.

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