Biochimica et Biophysica Acta, 1132 (1992) 17-25 17 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00 BBAEXP 92396 Enriched sources of Escherichia coli replication proteins. The dnaG primase is a zinc metalloprotein N. Patrick J. Stamford 1, Penelope E. Lilley and Nicholas E. Dixon Centre for Molecular Structure and Function, Research School of Chernistry, Australian National Unicersity, Canberra (Australia) (Received 13 January 1992) (Revised manuscript received 1 April 1992) Key words: DNA replication; Protein overproduction; Primase; Zinc finger; (E. coli) Primase, the product of the Escherichia coil dnaG gene, is the enzyme responsible for RNA primer synthesis on both template strands at replication forks during chromosomal DNA synthesis. The dnaG gene was modified by replacement of the natural ribosome-binding site with one complementary to the 3' end of 16S rRNA, and then inserted downstream of tandem bacteriophage A PR and PL promoters in the pUC9-derived vector pCE30. Following thermal induction of transcription, the resulting plasmid pPL195 directed synthesis of primase activity to levels corresponding to approx. 120000 molecules per cell. The overproduced protein was soluble and was readily purified in high yield (31 mg per I of culture). Purified primase was monomeric, was fully active in priming replication at the bacteriophage G4 complementary strand origin, and was shown to contain 0.92 + 0.08 g atom of tightly-bound zinc per mol of protein. Potential zinc-binding amino-acid residues near the N-terminus of the protein were identified. Although a mutant primase lacking 27 amino acid residues from the N-terminus was partly soluble, it was completely inactive. Introduction More than 20 separate polypeptides act concertedly to replicate the chromosome of the bacterium Es- cherichia coli [1]. Although there is now an adequate understanding of the separate roles of each of the proteins in the experimentally-separable processes of initiation of replication at the chromosomal origin of replication (oriC), of DNA synthesis at replication forks, and of termination of replication, there is yet little information concerning the chemical mechanisms that underlie the multitude of protein-protein and pro- tein-nucleic acid interactions that occur during these events [1]. As a first step toward understanding these interactions in greater detail, it was necessary to use available genes to construct greatly enriched sources for isolation of the proteins in quantities sufficient for chemical and structural studies. In the present paper, we focus on improved overproduction, purification and some properties of the dnaG primase. Correspondence to: N.E. Dixon, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia. Present address: University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, UK. Abbreviations: LB, Luria-Bertani medium; SS, single-strand(ed). The E. coli primase is a 65.5 kDa DNA-dependent RNA polymerase. It is the enzyme responsible for synthesis of short RNA primers for DNA synthesis both on the leading strand at oriC [2] and, in associa- tion with the dnaB helicase and other components of a primosome [3], for repeated reinitiation on the lagging strand at replication forks [1]. It is encoded by the dnaG gene [4] located in the chromosome within a complex operon that includes genes (rpsU and rpoD) that encode proteins required for protein and RNA synthesis (ribosomal protein $21 and the ~ subunit of RNA polymerase, respectively). Regulation of expres- sion of the operon has been studied extensively [5-7], provoked in part by the observation that dnaG, the second gene, is expressed at a much lower level than the other two. Log-phase E. coli cells contain only 50 to 100 molecules of primase [1]. The dnaG gene has been isolated and its nucleotide sequence has been determined [5,8]. Construction of a plasmid, pRLM61, that directed approx. 100-fold overproduction of primase was re- ported in 1982 [9]. From this enriched source, it has been possible to isolate significant quantities of the enzyme, and this has facilitated studies of its function in reconstituted in vitro replication reactions [2,10-15]. Such quantities were not sufficient for our work, and pRLM61 does not provide the flexibility of manip-