Proposed translocation cycle of the restriction-modification system EcoR124I

V. Bialevich^1,2, K. Shamayeva^1,2, D. Sinha^1,2, A. Guzanova³, M. Weiserova³, E. Csefalvay¹,
J. Carey^1,4 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

⁴Department of Chemistry, Princeton University, New Jersey, USA

Type I R-M systems are composed of three different subunits: one HsdS subunit is required for identification of target sequence and anchoring of enzyme complex on DNA; two HsdM subunits in MTase complex serve for host genome modification accomplishing protective function against self-degradation; two HsdR (or motor) subunits take part in ATP-dependent translocation and consequent cleavage of dsDNA [1, 2].

The crystal structure of the 120 kDa HsdR subunit of the Type I R-M system EcoR124I in complex with ATP was recently reported by our group [3]. 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. The helical domain resembles HsdM and has strong interactions with helicase 2 domain [3].

Molecular mechanism of dsDNA translocation, cleavage and ATP hydrolysis has not been yet structurally investigated. Here we propose a translocation cycle of the restriction-modification system EcoR124I based on analysis of available crystal structures of superfamily 2 helicases, strutural modeling and complementary biochemical characterization of mutations introduced in sites potentially inportant for translocation in the HsdR motor subunit.

We gratefully acknowledge support from the Czech Science Foundation (project number GACR P207/12/2323).

1. Murray, N. E. Microbiology and Molecular Biology Reviews, 2000, Vol. 64, 412–434.

2. Dryden et al., Nucl. Acids Res., 2001, Vol. 29, 3728-3741.

3. Lapkouski M. et al, Nat. Struct. & Mol.Biol, 2009, 16, 94.