How can stalling of translocation activate the endonuclease in the type I restriction-modification system EcoR124I

K. Shamayeva^1,2, V. Bialevich^1,2, A. Samad^1,2, T. Baikova^1,2, A. Guzanova³, M. Weiserova³, L. Csefalvay^1,2, 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 provide a bacterial defence mechanism against foreign DNA. Hemi-methylated host DNA is fully methylated at specific sequences by a methyltransferase, thus protecting its DNA from restriction by the accompanying endonuclease. Foreign DNA is unmethylated at these sites and is cleaved [1, 2].

Type I restriction endonucleases consist of three different subunits: methylase (M), restriction (R) and specificity (S) encoded by the hsdM, -R and -S genes, where hsd=Host Specificity for DNA [3]. Together, they form an intriguing, multifunctional complex which can either restrict or modify DNA. Here, the mode of action of the complex is dictated by the methylation state of the recognition sequence [4]. A fully methylated site results in no action being taken and in enzyme dissociation; hemi-methylated target sequences direct the enzyme into a protective methylation mode producing fully methylated DNA, while unmethylated DNA shifts the enzyme into a destructive (and protective) restriction mode. It is in this protective mode that type I enzymes restrict foreign DNA and thereby maintain the integrity of the host genome [3].

Molecular mechanism of dsDNA translocation, cleavage and ATP hydrolysis has not been jet structurally investigated. Crystal structure of the 120 kDa motor HsdR protein of the Type I EcoR124I nuclease in complex with ATP was recently reported by our group, and it is the first know structure of the motor subunit throughout group of Type I restriction enzymes. This structure characterization, among others, disclose unexpected endonuclease domain contributing to ATP binding by Lys220 residue. This contact could potentially couple endonuclease and translocase functions [5].

To more clearly understand correlation of ATPase and restriction activities of the type I amino acid residues from sequence-variable region around Lys220 have been chosen which could be potentially engaged in conformational changes that occur once translocation is stalled and a signal is transmitted to the endonuclease. Those residues have been replaced by site-direct mutagenesis and the influence of the replacement is monitored using in vitro ATPase activity assays of the whole pentameric enzyme complex and is tested by restriction assay in vitro and in vivo.

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[3] Murray,N.E. Microbiol. Mol. Biol., Rev., 2000, 64, 412–434.

[4] Yuan,R., Bickle,T.A., Ebbers,W. and Brack,C. Nature, 1975, 256, 556–560.

[5] Lapkouski M., Panjikar S., Janscak P., Kuta Smatanova I., Carey J., Ettrich R., Csefalvay E. 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).