Crystal and Solution Structures of the Helicase-binding Domain of Escherichia coli Primase

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 DnaB 6 . Determination of the 2.8-Å 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 primasehelicase complex at replication forks.

Essential Biological Processes of an Emerging Pathogen: DNA Replication, Transcription, and Cell Division in Acinetobacter spp

“…Bacterial primases contain three distinct domains: a zinc-binding domain at the N terminus, a central RNA polymerase (RNAP) domain, and a C-terminal helicasebinding domain that is structurally related to the N-terminal domain of DnaB (51,78,191,237,241,258). The structural basis for the primosome interaction was revealed recently in crystal structures of the complex between DnaB and the Cterminal domain of DnaG from Geobacillus stearothermophilus (12).…”

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

“…One strand (the leading strand) is replicated continuously, while the other (lagging) strand is synthesized discontinuously in a series of (Okazaki) fragments. The replicative RNA-priming enzyme, DnaG primase (49), is recruited by DnaB for the priming of each new fragment on the discontinuous strand (133). The single-stranded sections that result from helicase action are coated with single-stranded DNAbinding protein (SSB).…”

“…Each DnaB monomer (471 residues) is made up of two domains linked by a region that may function as a flexible hinge (129). The Nterminal domain, comprising residues 24 to 136 (123), undergoes a monomer-dimer equilibrium in solution (171), is dimeric in the crystalline state (46), and appears to participate in interaction with the DnaG primase (21,29,133). The larger C-terminal domain (containing residues from about 170 to 471) is a hexamer and bears the ATPase and DNA-binding sites (21,129).…”

In the Bacillus stearothermophilus DnaB-DnaG Complex, the Activities of the Two Proteins Are Modulated by Distinct but Overlapping Networks of Residues

“…A distinct C-terminal helicase-interacting domain of DnaG (known as P16) mediates this interaction structurally and functionally (2,21). The recently determined structures of P16 from Bacillus stearothemophilus and Escherichia coli DnaG revealed that it consists of two subdomains, a six-helix bundle (subdomain C1) that is essential for stimulation of DnaB activity and a helical hairpin (subdomain C2) that mediates binding to DnaB (13,19). DnaG interacts with a linker region that joins the N-and C-terminal domains of DnaB and induces the formation of threefold symmetric rings with mainly three DnaG molecules interacting with one DnaB hexamer (20).…”