LraI fromLactococcus raffinolactisBGTRK10-1, an Isoschizomer ofEcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity

Abstract

Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity.Lactococcus raffinolactisBGTRK10-1 expressesLraI Type II restriction-modification enzyme, whose activity is similar to that shown forEcoRI;LraI methyltransferase protects DNA fromEcoRI cleavage. The gene encodingLraI endonuclease was cloned and overexpressed inE. 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 ofLraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence forLraI restriction enzyme was determined as 5′-G/AATTC-3′, indicating thatLraI restriction enzyme is an isoschizomer ofEcoRI. In the reaction buffer with a lower salt concentration,LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 5′-A/AATTC-3′, leaving the same sticky ends on fragments asEcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin ofLraI restriction-modification system with previously describedEcoRI-like restriction-modification systems.