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

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

doi:10.1074/jbc.273.34.21608

Abstract

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.

Highlights

DNA polymerase ␣-primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions
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
The discussion concerning the function of the primase subunits was revitalized when the cloning and subsequent expression of pol-prim subunits from different organisms led to a controversy whether p48 alone can synthesize the first dinucleotide and elongates it or whether it has only elongation activity and requires p58 for the synthesis of the first dinucleotide [22,23,24,25,26]

Summary

Introduction

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. 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. DNA polymerase ␣ and primase are essential proteins of the cellular DNA replication machinery They are important components of the initiation complex and play a central role in leading and lagging strand synthesis [1,2,3,4,5,6,7]. The bacterial and baculovirus expressed proteins could be assembled in vitro and formed a four-subunit pol-prim complex that is active in DNA replication

Methods

Results

Conclusion