Preparation of HsdR subunit of EcoAI endonuclease

 

Tatsiana Baikova, Ivana Kuta Smatanova, Eva Csefalvay

 

Department of Structure and Function of Proteins, Institute of Systems Biology and  Ecology, Academy of Sciences of the Czech Republic; and Institute of Physical Biology, University of South Bohemia in Ceske Budejovice, Zámek 36, CZ-373 33 Nove Hrady, Czech Republic

 

 

Restriction enzymes (RE) are components of bacterial restriction-modification (R-M) systems that serve to protect the cells against bacteriophage infection, because the incoming foreign DNA is endonucleotically cleaved by the restriction enzyme if it contains the enzyme's recognition sequence. R-M systems can be divided into several different types according to subunit composition and cofactor requirements. RE Type I are hetero-oligomeric enzyme compose of 5 subunits, which are responsible for specificity (HsdS), methylation activity (HsdM), ATP-dependent DNA translocation and endonuclease activity (HsdR).  Although RE Type I has been extensively characterized biochemically, there is not much direct structural information available about particular subunits. Structure of HsdR subunit of EcoR124I (RE TypeIC) has been described recently by our group (1). Due to differences in length and composition in primary sequence of HsdR of particular RE Type I, we decide to structurally characterize HsdR subunit of EcoAI endonuclease belonging to the type IB family. Comparison of structural information supposes to give us more complex information about catalytical domains and function of these peliculiar endonucleases. Our project described here is aimed to produce enzyme in amount and purity sufficient for crystallization.

1. Lapkouski M, Panjikar S, Janscak P, Smatanova IK, Carey J, Ettrich R, Csefalvay E. Structure of the motor subunit of type I restriction-modification komplex EcoR124I. Nat Struct Mol Biol. (2009) Jan;16(1):94-5. Epub 2008 Dec 14.