Lra I from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of Eco RI, Exhibits Ion Concentration-Dependent Specific Star Activity.

Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. Lactococcus raffinolactis BGTRK10-1 expresses Lra I Type II restriction-modification enzyme, whose activity is similar to that shown for Eco RI; Lra I methyltransferase protects DNA from Eco RI cleavage. The gene encoding Lra I endonuclease was cloned and overexpressed in E. coli . Purified enzyme showed the highest specific activity at lower temperatures (between 13°C and 37°C) and was stable after storage at -20°C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity of Lra I restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for Lra I restriction enzyme was determined as 5'-G/AATTC-3', indicating that Lra I restriction enzyme is an isoschizomer of Eco RI. In the reaction buffer with a lower salt concentration, Lra I exhibits star activity and specifically recognizes and cuts another alternative sequence 5'-A/AATTC-3', leaving the same sticky ends on fragments as Eco RI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of Lra I restriction-modification system with previously described Eco RI-like restriction-modification systems.