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.