FUNCTIONAL COUPLING Of DNA DUPLEX TRANSLOCATION TO ITS CLEAVAGE IN A TYPE I RESTRICTION ENZYME
R. Ettrich1, 2, M. Lapkouski1, A. Guzanova3 , L. Csefalvay1, T. Baikova1, 2, E. Dellapenna4, M. Khabiri1,2, I. Shevelev4, P. Janscak5,6, S. Panjikar7, J. Carey 1,7, Marie Weiserova3 , I. Kuta Smatanova1, 2, E. Csefalvay 1, 2
1Institute of Nanobiology and Structural Biology of ISBE, Academy of Sciences of the Czech Republic, 37333 Nove Hrady, Czech Republic, 37333 Nove Hrady, Czech Republic
2University of South Bohemia in České Budějovice, 37333 Nove Hrady, Czech Republic
3Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic
4Department of Chemistry, Princeton University, Princeton, New Jersey 08544-1009, USA
5Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic
6Institute of Molecular Cancer Research, University of Zürich, Wintherthurerstrasse 190, CH-8057 Zürich, Switzerland
7EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D22603 Hamburg, Germany
The type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to pull thousands of basepairs through the enzyme before cleaving distant sites nonspecifically. Biochemical, biophysical, and molecular biological studies of their translocation and cleavage mechanisms offer a wealth of detail that has lacked a structural framework. The crystal structure of the motor subunit responsible for DNA translocation and cleavage by the type I enzyme EcoR124I, resolved at 2.6 A , shows a lysine residue on the endonuclease domain to contact N3 on the exposed edge of ATP bound at the helicase domains, potentially coupling endonuclease and translocase functions. Protein crystallography, point-mutations, in vivo and in vitro testing, in combination with computational modelling are used as tools to explain the coupling of endonuclease and translocase functions in Type I restriction-modification complexes in general.
1. Lapkouski M, Panjikar S, Janscak P, Kuta Smatanova I, Carey J, Ettrich R, Csefalvay E Nat Struct Mol Biol 16: 1. (2009) 94-95.
We gratefully acknowledge support from the Ministry of Education, Youth and Sports of the Czech Republic (MSM6007665808, LC06010), the Academy of Sciences of the Czech Republic (AVOZ60870520), the Grant Agency of the Czech Republic (Nos. P207/10/1934), and joint Czech - US National Science Foundation International Research Cooperation (ME09016 and INT03-09049), Additionally, T.B. and M.K. were supported by the University of South Bohemia, grant GAJU 170/2010/P.