Identification of a restriction endonuclease (SacC1) from Saccharomyces cerevisiae

摘要

SacC1 is a novel restriction endonuclease from?Saccharomyces cerevisiae?that recognizes the palindromic sequence 5’CTCGAC3’ cleaving both DNA strands upstream and downstream of its recognition sequence and makes a staggered cut at the distance of five bases from the recognition sequence on the upper strand and at the seventh base on the complementary strand. It shares similar characteristics with Sac I from?Streptomyces achromogenes?as well as Sst1 from Streptomyces Stanford and Psp124B1 from Pseudomonas species. It has been purified by ammonium sulphate precipitation, dialysis, and gel filtration using phosphocellulose, DEAE-cellulose and Sephadex G-100 with an optimal pH range (7.5-8.5), active at 37°C and dependent on Mg2+?or Mn2+?which increases its activity by 4- and 2-folds, respectively, while other cations decrease its activity to some extents. Cleavage on both sides of the recognition sequence is characteristic of Type IIB systems but all IIB enzymes studied so far have been found to recognize discontinuous sites and a distinctive subunit/domain organization that is not present in the SacC1 enzyme. There are similarities between SacC1 and other homing endonucleases belonging to the LAGLIDADG family such as a requirement for Mg2+?(or Mn2+) for cleavage to take place, optimal activity at alkaline pH and stimulation of the reaction by moderate concentrations of the monovalent cation.