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

• Published in: BioMed research international Vol. 2018; p. 5657085
• Main Authors: Miljkovic, Marija, Malesevic, Milka, Filipic, Brankica, Vukotic, Goran, Kojic, Milan
• Format: Journal Article
• Language: English
• Published: United States 2018
• ISSN: 2314-6141

Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. BGTRK10-1 expresses I Type II restriction-modification enzyme, whose activity is similar to that shown for RI; I methyltransferase protects DNA from RI cleavage. The gene encoding I endonuclease was cloned and overexpressed in . 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 I restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for I restriction enzyme was determined as 5'-G/AATTC-3', indicating that I restriction enzyme is an isoschizomer of RI. In the reaction buffer with a lower salt concentration, I exhibits star activity and specifically recognizes and cuts another alternative sequence 5'-A/AATTC-3', leaving the same sticky ends on fragments as RI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of I restriction-modification system with previously described RI-like restriction-modification systems.