A Dna Mimic Caught In The Act: 3D Electron Microscopy Shows Ecoki Methyltransferase In Complex With The T7 Antirestriction Protein Ocr.

Abstract

Type I DNA restriction-modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of the DNA target or translocation, followed by cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein Ocr. Ocr mimics DNA with a pseudo-helical arrangement of charges, and is bent at a similar angle to that predicted for target DNA. We present a 3D density map generated by negative stain electron microscopy of the central core of the restriction complex, M.EcoKI M2S1 methyltransferase, bound to dimeric Ocr. Single particle analysis was carried out in IMAGIC and EMAN and resulted in a 3D reconstruction at ∼18 A resolution. An atomic model of all 5 subunits was generated by automated docking and homology modelling. This was computationally fitted into the EM density, giving excellent agreement. Ocr binds through the center of the M.EcoKI complex, spanning the two DNA recognition sites and matching the path predicted for its substrate DNA. We also present a complete atomic model of M.EcoKI in complex with its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of published experimental data on EcoKI spanning 40 years.