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DNA primases catalyse the synthesis of the short RNA primers on single stranded (ss) DNA templates that are used by DNA polymerase to initiate the synthesis of Okazaki fragments on the lagging strand. Primase is an essential enzyme in all well characterised systems of DNA replication. The domain structure of bacterial DNA primase has been studied using limited proteolysis. These studies indicated that there are three functional domains in the protein. The N-terminal 12 KDa fragment contains a zinc-binding motif. The central fragment of 37 KDa contains a number of conserved sequence motifs that are characteristic of primases, including the so-called "RNA polymerase (RNAP)-basic" motif that shows homology with equivalent motifs in prokaryotic and eukaryotic RNAP large subunits. This suggects that primases might share a common structural mechanism with RNAP. The C-terminal domain of approximately 150 residues is the part of the protein responsible for interaction with the replicative helicase, DnaB, at the replication fork. A comparison of three bacterial primases and four bacteriophage primases (T7, T3, T4 and P4) has demonstrated the evolutionary conservation of five sequence motifs including the zinc-binding motif and the RNAP-basic motif described above. Two of the other motifs were proposed to be involved in Mg²⁺-mediated NTP binding motif, the motifs are all within the central part of the primase sequence, which probably constitutes the catalytic core of the enzyme.

Zinc-binding motifs are commonly shared by proteins that are involved in RNA or DNA transactions, and frequently mediate sequence-specific protein-DNA interactions. The various zinc-binding protein families exhibit considerable structural variation. To date, six major families have been charaterised, with analogous sequences being found in a variety of genes involved in DNA or RNA transactions. These include eukaryotic transcription factors TFIIS and TFIIB, subunits of RNAP II, general transcription factors, DNA polymerases and some bacterial DNA-repair proteins.

In order to improve our understanding of the interaction between DNA primase and its ssDNA template, the primase from Bacillus stearothermophilus was chosen as a target for crystallography. This protein was chosen because proteins from thermostable organisms often have a great tendency to crystallise. We cloned the dnaG gene, which encodes the DNA primase from B. stearothermophilus, and overexpressed the protein in E. coli. This primase is active in a general priming assay (K. Marians, unpublished data). We have crystallised the N-terminal fragment of DNA primase from B. stearothermophilus, which contains the zinc-binding motif, and determined its structure at 1.7 Å resolution. The structure confirms that this domain is a member of the zinc ribbon family and comparison with other related proteins suggests how this portion of primase might interact with the ssDNA template.

(a)The electron density map at 1.7Å resolution showing the zinc ion bound to its four ligands. The 2Fo-Fc (yellow) and Fo-Fc (red) maps are contoured at 1 and 2 sigma respectively. (b) The position of the zinc ion and its four ligands.