The influence of the C-terminal helical domain on ATPase activity in the restriction-modification complex EcoR124I

V. Bialevich^1,2, K. Shamayeva^1,2, A. Guzanova³, M. Weiserova³, E. Csefalvay^1,2 and R. Ettrich^1,2

¹Institute of Nanobiology and Structural Biology of GCRC, AS CR, 37333 Nove Hrady, Czech Republic

²University of South Bohemia in České Budějovice, 37333 Nove Hrady, Czech Republic

³Institute of Microbiology, AS CR, Vídeňská 1083, 142 20, Praha 4

Restriction-modification enzymes (R-M) protect bacteria from infections by viruses, and it is commonly accepted as being their major role in nature, thus they function as the main players constituting microbial immune systems [1]. The phenomenon of restriction, identified first for type I R-M systems, laid the foundations for modern molecular biology, and, eventually, led to the discovery of widely used in DNA cloning techniques and highly commercialized type II restriction enzymes. The classical R-M systems of Escherichia coli K-12 and E.coli B were first to be discovered by Bertani and Weigle back to 1953 [2]. The classical type I R-M enzymes of E. coli K-12 (EcoKI) and B (EcoBI) were not only the first to be detected but also the first to be purified [3, 4].

Type I R-M enzymes are large, multifunctional macromolecular complexes composed of three different subunits: HsdS, HsdM and HsdR [5]. The activities of the complex of all three subunits include ATP-dependent DNA translocation, DNA cleavage and methylation [6-9].

HsdR is organized into four approximately globular structural domains in nearly square-planar arrangement: the N-terminal endonuclease domain, the recA-like helicase domains 1 and 2 and the C-terminal helical domain. The near-planar arrangement of globular domains creates prominent grooves between each domain pair. The two helicase-like domains form a canonical helicase cleft in which double-stranded B-form DNA can be accommodated without steric clash. A positively charged surface groove proceeds from the helicase cleft and continues between the helical and endonuclease domains where it passes over the cleavage site recessed slightly from the surface. The helical domain resembles HsdM and has strong interactions with helicase 2 domain [10].

In the present work we test mutations, which could be essential for helical-helicase 2 domains interactions, by using a combination of site-directed mutagensis and in vivo and in vitro restriction activity assays. Absence of these interactions influences either subunit assembly, rotation of helicase 2 domain relative to helicase 1 domain, loading of dsDNA in the helicase cleft or DNA translocation and following restriction.

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[9] McClelland et al., J. Mol. Biol. 2005, 348, 895–915.

[10] Lapkouski M. et al, Nat. Struct. & Mol.Biol. 2009, 16, 94.

We gratefully acknowledge support from the Czech Science Foundation (project number GACR P207/12/2323), and the Grant Agency of the University of South Bohemia (grant no. 170/2010/P). Some computations were performed in MetaCentrum SuperComputer facility.