Purification and Initial Imaging of the type I DNA Restriction-Modification Enzyme EcoR124I by Electron Microscopy

Ladislav Csefalvay¹, Daniel Nemecek², Eva Csefalvay¹, Rudiger Ettrich¹

¹Institute of Nanobiology and Structural Biology, Zamek 136, Nove Hrady, CZ

²Central European Institute of Technology–Masaryk University, Kamenice 5, Brno, CZ

Restriction enzymes protect prokaryotic cells from viral infection by recognition and degradation of invading DNA. These enzymes are typically coupled with specific modification enzymes that mark and protect cellular DNA from degradation by methylation. The type I DNA restriction-modification enzymes unite both functions in a large multisubunit complex [1, 2]. The prototype enzyme EcoR124I is composed of one HsdS subunit that binds to a specific sequence of DNA, two HsdM subunits that are responsible for methylation of the cellular DNA and two HsdR subunits that are required for restriction of the nonmethylated (invading) DNA. Basic quaternary structure of the whole R₂M₂S complex has been shown by negative-stain electron microscopy at low resolution [3]. Although high-resolution structures of the individual subunits have been determined by crystallography or molecular modeling, the current EM structure is insufficient for accurate fitting of the known atomic models and to uncover the mechanisms of EcoR124I assembly and function.

We used affinity chromatography and gel filtration to purify the R₂M₂S complex. Densitometry quantitative analysis of SDS-PAGE gels of purified complex indicated average stoichiometry of 1.6(R):2.0(M):1.1(S) suggesting that only about a half of the assembled complexes contained both HsdR subunits, likely because of significantly different binding affinity of the two HsdR subunits to the M₂S subcomplex (K_D1 0.6 nM vs. K_D2 190 nM).

The purified EcoR124I complex was negatively-stained in 2% uranyl acetate and imaged electron microscopy. Images of the negatively stained complex showed projections consistent with the previously determined structure of the EcoR124I complex in the open conformation [3]. The purified complex was also plunge-frozen in liquid ethane and imaged by cryo-electron microscopy. Micrographs were collected at 2-4 µm defocus using a FEI Tecnai F20 microscope. The micrographs show distinct particles of the presumed EcoR124I complex (~120 Å diameter), despite the relatively small size of the complete R₂M₂S complex (~450 kDa). Further, methods of single particle analysis [5] will be used to reconstruct the EcoR124I complex to high resolution that would then allow reliable fitting of the atomic models of the individual subunits.

[1] Youell, J. and Firman, K. (2008) Microbiol Mol Biol Rev., 72, 365.

[2] Janscak, P., Dryden, D.T.F., Firman, K. (1998) Nucleic Acids Res., 26, 4439.

[3] Kennaway,C.K., et al. (2012) Genes Dev., 26, 92.

[4] Mernagh DR, Janscak P, Firman K, Kneale GG. , Biol Chem. 1998 379, 497-503.

[5] Frank, J. (2006) “Three-Dimensional Electron Microscopy of Macromolecular Assemblies”, Edition 2, Oxford University Press, Inc., New York.