Identification of amino acid residues important for coupling ATPase and restriction activity in EcoR124I endonuclease
K. Shamayeva1,2, V. Bialevich1,2, L. Csefalvay1, R. Ettrich1,2
and E. Csefalvay1,2
1Institute of Nanobiology and Structural Biology of ISBE, AS CR, 37333 Nove Hrady,
2University of South Bohemia in České Budějovice, 37333 Nove Hrady, Czech Republic
Restriction-modification enzymes (R-M) protect bacteria from infections by viruses, and it is commonly accepted as being their major role in nature. Type I R-M enzymes are large, multifunctional macromolecular complexes composed of three different subunits: HsdS, HsdM and HsdR . The activity of the complex endonuclease is translocation DNA through enzyme complex tightly binds in specific sequence on DNA molecule. When translocation is stopped, the DNA molecule is cleaved nonspecifically outside of recognition site . The first crystal structure of the 120 kDa motor HsdR protein of the Type I EcoR124I nuclease in complex with ATP was recently reported . This structure discloses an unexpected contact of endonuclease domain with ATP by Lys220. Engagement of the endonuclease domain in ATP binding seems to be involved in the communication of the ATP-dependent translocase with the endonuclease, potentially coupling dsDNA translocation and cleavage.
To investigate the underpinning molecular mechanism of coupling ATP-dependent DNA translocation and DNA cleavage and the communication pathway through the motor subunit, we selected residues from the sequence variable region nearby Lys220, which could be potentially engaged in conformational changes that occur once translocation is stalled and a signal is transmitted to the endonuclease. The influence of the replacement of these selected residues to either neutral Ala or eventually to charged Arg on translocation is monitored using in vitro ATPase activity assays of the whole pentameric enzyme complex. We demonstrate that although in close proximity to the ATP binding pocket, none of the selected residues significantly alters ATPase activity, thus leaving the ATPase fully intact. Alteration of endonuclease activity of these selected residues is reported and discussed in molecular terms.
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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, K.S. was supported by the University of South Bohemia, grant GAJU 170/2010/P.