Abstract
During bacterial DNA replication, the DnaG primase interacts with the hexameric DnaB helicase to synthesize RNA primers for extension by DNA polymerase. In Escherichia coli, this occurs by transient interaction of primase with the helicase. Here we demonstrate directly by surface plasmon resonance that the C-terminal domain of primase is responsible for interaction with DnaB6. Determination of the 2.8-angstroms crystal structure of the C-terminal domain of primase revealed an asymmetric dimer. The monomers have an N-terminal helix bundle similar to the N-terminal domain of DnaB, followed by a long helix that connects to a C-terminal helix hairpin. The connecting helix is interrupted differently in the two monomers. Solution studies using NMR showed that an equilibrium exists between a monomeric species with an intact, extended but naked, connecting helix and a dimer in which this helix is interrupted in the same way as in one of the crystal conformers. The other conformer is not significantly populated in solution, and its presence in the crystal is due largely to crystal packing forces. It is proposed that the connecting helix contributes necessary structural flexibility in the primase-helicase complex at replication forks.
Highlights
All organisms replicate DNA in a semidiscontinuous fashion [1]
surface plasmon resonance (SPR) Analysis of DnaB6 Binding to Immobilized DnaG and DnaG-(1– 433)—The interactions of DnaB6 with immobilized full-length DnaG (65,572 Da) and a truncated form lacking the C-terminal domain (DnaG-(1– 433), 48889 Da; termed p49) were first analyzed by SPR (BIAcore)
Because most of the immobilized primase was inactive, presumably because the DnaB6-binding site was occluded during covalent immobilization, it was not possible to determine the stoichiometry of the complex with this surface
Summary
Protein Purification—Overproduction of DnaG, DnaG-(1– 433), and DnaG-C was achieved in E. coli strains containing plasmid derivatives of vector pCE30, pMA200U [28], or pND706 [29], respectively, with appropriately constructed genes under control of thermoinducible tandem bacteriophage pR and pL promoters. To prepare plasmid pCP806 (dnaG-(1– 433)ϩ), the dnaG gene from pPL195 was transferred to the phagemid vector pMA200U, and the singlestranded form was used as template for oligonucleotide-directed mutagenesis [31] to replace codon 434 of dnaG with a TAA stop codon followed by an EcoRI site. DnaB6 was purified using strain AN1459/pPS562 [6]. Plasmid pPS562 contains a synthetic dnaCB operon under transcriptional control of the tandem pR and pL promoters and directs high level overproduction of equimolar amounts of DnaB and DnaC proteins. SPR Measurements—A BIAcore 2000 instrument (Biacore AB, Uppsala, Sweden) was used to study the interactions of DnaB with DnaG as TABLE I MAD phasing statistics (resolution range 50 to 2.8 Å)
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