Primase activity of human DNA polymerase alpha-primase. Divalent cations stabilize the enzyme activity of the p48 subunit.
DNA polymerase alpha-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 alpha-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.
Institut für Molekulare Biotechnologie, Beutenbergstrasse 11, D-07745 Jena, Germany.
A
A
Schneider
R W
RW
Smith
A R
AR
Kautz
K
K
Weisshart
F
F
Grosse
H P
HP
Nasheuer
D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism*; D002417:Cattle; D004256:DNA Polymerase I / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D004261:DNA Replication; D004926:Escherichia coli; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011066:Poly C / Q000378:metabolism; D011071:Poly T / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D013539:Simian virus 40 / Q000235:genetics; D013698:Templates, Genetic
D002413:Cations, Divalent; D011071:Poly T; C023826:poly(dC); D011066:Poly C; D008345:Manganese; D019915:DNA Primase; D004256:DNA Polymerase I; D008274:Magnesium; D011188:Potassium