Abstract
DnaA forms a homomultimeric complex with the origin of chromosomal replication (oriC) to unwind duplex DNA. The interaction of the DnaA N terminus with the DnaB helicase is crucial for the loading of DnaB onto the unwound region. Here, we determined the DnaA N terminus structure using NMR. This region (residues 1–108) consists of a rigid region (domain I) and a flexible region (domain II). Domain I has an α-α-β-β-α-β motif, similar to that of the K homology (KH) domain, and has weak affinity for oriC single-stranded DNA, consistent with KH domain function. A hydrophobic surface carrying Trp-6 most likely forms the interface for domain I dimerization. Glu-21 is located on the opposite surface of domain I from the Trp-6 site and is crucial for DnaB helicase loading. These findings suggest a model for DnaA homomultimer formation and DnaB helicase loading on oriC.
Highlights
Domain I Dimerization—In this study, we have described the structure of DnaA domain I
A recently published crystal structure of the K homology (KH) domain in PCBP2 revealed that it forms homodimers [39]
Based on PCBP2 dimer structure and our experimental results, we have proposed a structural model of dimerization between domain I monomers (Fig. 3D)
Summary
Protein Preparation of the DnaA N terminus—A 5Ј-region of the wild-type dnaA corresponding to amino acid residues 1–128 was amplified by PCR. DnaA was incubated at 30 °C for 30 min in buffer (25 l) containing 20 mM Tris-HCl (pH 7.5), 0.1 mg/ml bovine serum albumin, 8 mM dithiothreitol, 10 mM Mg(OAc) 125 mM potassium glutamate, 2 mM ATP, 2.32 g of single strand-binding protein (SSB), 2.5 ng of HU protein, 150 ng of DnaB, 92 ng of DnaC, 180 ng of DNA gyrase A subunit, 225 ng of DNA gyrase B subunit, and 200 ng of M13KEW101 minichromosome. The indicated amounts of the isolated DNA-loaded clamps were incubated at 30 °C for 20 min in buffer containing 20 mM Tris-HCl (pH 7.5), 10% glycerol, 8 mM dithiothreitol, 0.01% Brij-58, 8 mM Mg(OAc) 120 mM potassium glutamate, 2 mM ATP, 0.1 mg/ml bovine serum albumin, 56 fmol of the C-terminally His-tagged Hda, and [␣-32P]ATP-DnaA (0.25 pmol). Nucleotides bound to DnaA were recovered by filter retention, separated by thin-layer chromatography, and quantified by a BAS2500 bioimaging analyzer (Fuji Film)
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