PDBsum entry 1zt2

Replication, transferase

PDB id: 1zt2

Contents

Protein chains

327 a.a.

207 a.a.

Ligands

SO4 ×39

Metals

_ZN ×2

Waters ×12

References listed in PDB file

Key reference

Title

Structure of the heterodimeric core primase.

Authors

S.H.Lao-Sirieix, R.K.Nookala, P.Roversi, S.D.Bell, L.Pellegrini.

Ref.

Nat Struct Mol Biol, 2005, 12, 1137-1144. [DOI no: 10.1038/nsmb1013]

PubMed id

16273105

Note In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above were identified by an automated search of PubMed on title and author names, giving a percentage match of 85%.

Abstract

Primases are DNA-dependent RNA polymerases that synthesize the oligoribonucleotide primers essential to DNA replication. In archaeal and eukaryotic organisms, the core primase is a heterodimeric enzyme composed of a small and a large subunit. Here we report a crystallographic and biochemical analysis of the core primase from the archaeon Sulfolobus solfataricus. The structure provides the first three-dimensional description of the large subunit and its interaction with the small subunit. The evolutionary conservation of amino acids at the protein-protein interface implies that the observed mode of subunit association is conserved among archaeal and eukaryotic primases. The orientation of the large subunit in the core primase probably excludes its direct involvement in catalysis. Modeling of a DNA-RNA helix together with structure-based site-directed mutagenesis provides insight into the mechanism of template DNA binding and RNA primer synthesis.

Figure 4.
Figure 4. Analysis of the PriS-PriL interface. (a) Hydrophilic interactions at the PriS-PriL interface. Hydrogen bonds are yellow dashed lines. The carbonyl groups of Asp162 and Asp163 at the C terminus of PriS helix 4 are hydrogen-bonded to the main chain amides of PriL residues Lys165 and Gly166. The side chains of Asp162 and Asp163 further interact electrostatically with Arg224 and Arg227. (b) Structure-based yeast two-hybrid analysis of the Sso PriS-PriL interface. Single and double mutations in PriS and PriL disrupt the interaction between the core primase subunits. Control plate: -Leu, -Trp; selective plates: -Leu, -Trp, -His and the more stringent -Leu, -Trp, -His, -Adenine. Numbered samples are identified in Table 1.

Figure 6.
Figure 6. Interaction of the Sso core primase with DNA template and RNA primer. (a) The zinc-binding motif of archaeal primases. The small subunits of Sso, Pho (PDB entry 1V33) and Sis (PDB entry 1RNI) primases were superimposed. The Sso PriS-prim is shown as a molecular surface. The zinc-binding motifs of Sso (green), Pho (yellow) and Sis (pink) are narrow tubes and their zinc atoms are spheres. (b) Comparison of the enzymatic activity of wild-type Sso core primase (Pri-WT) with that of the RR and SNG mutants (Pri-RR and Pri-SNG, respectively). Experiments were performed as for Figure 1c. DN, dinucleotide product. (c) Comparison of the quantities of product synthesized by Pri-WT and the RR and SNG mutants. Experiments were performed as for Figure 1c, with 1.2 M primase concentration. The radiolabeled products were quantified by filter binding and liquid scintillation counting. Each bar represents the average of five independent values with s.e.m. indicated. (d) Model of the Sso core primase-DNA template-RNA primer complex. The protein component of the complex is depicted as a molecular surface. The phosphate backbones of DNA and RNA are orange and cyan tubes, respectively. The proposed trajectory of the template DNA across the surface of the core primase is drawn. The position of PriS-Zn and the putative position of PriL-CTD are indicated by solid and dashed circles, respectively. The side chains of basic residues on and near PriL 5 are blue. The positions of PriL residues Arg84 and Arg85 are indicated.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2005, 12, 1137-1144) copyright 2005.