PDBsum entry: 3b39

Transferase/DNA

PDB id: 3b39

Contents

Protein chains

317 a.a. *

DNA/RNA

Waters ×146

* Residue conservation analysis

PDB id:

3b39

Name:

Transferase/DNA

Title:

Structure of the dnag primase catalytic domain bound to ssdn

Structure:

DNA primase. Chain: a, b. Fragment: RNA polymerase domain. Engineered: yes. DNA (5'- d( Dcp Dap Dap Dap Dgp Dcp Dcp Dap Dap Dap Dap Dgp Dgp Dap Chain: c, d. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 83333. Strain: k12. Gene: dnag, dnap, parb. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes

Resolution:

2.35Å R-factor: 0.214 R-free: 0.254

Authors:

J.E.Corn,J.G.Pelton,J.M.Berger

Key ref:

J.E.Corn et al. (2008). Identification of a DNA primase template tracking site redefines the geometry of primer synthesis. Nat Struct Mol Biol, 15, 163-169. PubMed id: 18193061 DOI: 10.1038/nsmb.1373

Date:

19-Oct-07 Release date: 15-Jan-08

Protein chains

P0ABS5  (PRIM_ECOLI) -  DNA primase

Seq: 581 a.a.

Struc: 317 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 

• Biological process: DNA replication, synthesis of RNA primer (1 term)
• Biochemical function: nucleotidyltransferase activity (2 terms)

DOI no: 10.1038/nsmb.1373

Nat Struct Mol Biol 15:163-169 (2008)

PubMed id: 18193061

Identification of a DNA primase template tracking site redefines the geometry of primer synthesis.

J.E.Corn, J.G.Pelton, J.M.Berger.

ABSTRACT

Primases are essential RNA polymerases required for the initiation of DNA replication, lagging strand synthesis and replication restart. Many aspects of primase function remain unclear, including how the enzyme associates with a moving nucleic acid strand emanating from a helicase and orients primers for handoff to replisomal components. Using a new screening method to trap transient macromolecular interactions, we determined the structure of the Escherichia coli DnaG primase catalytic domain bound to single-stranded DNA. The structure reveals an unanticipated binding site that engages nucleic acid in two distinct configurations, indicating that it serves as a nonspecific capture and tracking locus for template DNA. Bioinformatic and biochemical analyses show that this evolutionarily constrained region enforces template polarity near the active site and is required for primase function. Together, our findings reverse previous proposals for primer-template orientation and reconcile disparate studies to re-evaluate replication fork organization.

Selected figure(s)

Figure 1.
(a) Nucleic acid (sticks) occupies a binding groove formed by two -hairpins (green). Conserved residues previously known to be involved in primer synthesis are shown as gray sticks. Inset left, simulated annealing omit mF[o] – dF[c] difference map contoured at 3 , with the final modeled conformation of the ssDNA shown in yellow. Inset right, side view showing how the -hairpins buttress ssDNA. (b) The template-binding groove is the most strongly basic (blue) feature of primase's surface. The active site, which binds divalent metals, is highly acidic (red) and located opposite the basic ridge.

Figure 2.
(a) Contact map of primase's interaction with DNA as seen for the two molecules in the asymmetric unit. Hydrogen bonds or electrostatic interactions are indicated as dotted lines with atomic distances noted; solid vertical bars indicate van der Waals contacts. Conserved residues are shown in bold and evolutionarily coupled residues (Fig. 3) are shown in italics. Interactions within hydrogen-bonding distance are colored black; those that are too distant to allow hydrogen bonds are gray. (b) Close-up of nucleic acid-protein contacts within the template binding groove (molecule A above, molecule B below). See also Supplementary Movie 1.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2008, 15, 163-169) copyright 2008.

Figures were selected by an automated process.