Characterization of Type 2 Restriction Endonucleases (Hpy51) from Helicobacter pylori Strain 51

Cho, Myung Je; Park, Jeong Uck; Jeon, Beong Sam; Pack, Jeong Won; Byun, Eun Young; Lee, Sun Kyung; Park, Ye Hyoung; Song, Jae Young; Lee, Woo Kon; Baik, Seung Chul; Choi, Yeo Jeong; Jung, Seun Ae; Choe, Mi Young; Choi, Sang Haeng; Ko, Gyung Hyuck; Youn, Hee Shang; Rhee, Kwang Ho

Abstract

This study describes the purification and characterization of type II restriction endonuclease of Helicobacter pylori in order to understand the DNA restriction and modification of H. pylori. H. pylori cell extract was subjected to polyethyleneimine treatment, salt precipitation, heparine-sepharose column chromatography, and fast protein liquid chromatography (FPLC) using Resource Q column and Mono Q column to purify the type II restriction endonuclease. Hpy51-I was characterized to recognize the sequneces 5`-GT(G/C)AC-3`, yielding 5-base 5` protruding ends. The restriction sequence was identical to that of Tsp 45 I. The enzyme exhibited its maximal activity in the presence of 10-20 mM LaCl, but was inhibited completely in the presence of more than 80 mM NaCl. The enzyme showed its maximal activity in the presence of 1-10 mM MgC1(2). The optimal pH and temperature for enzyme activity was pH 9.0 and 37 degrees C, respectively. MnC1(2) could not substitute for MgC1(2) in reaction mixture. And addition of j3-mercaptoethanol and bovine serum albumin in reaction mixture led to loss of enzyme activity of Hpy51-I. The whole cell extract of H. pylori strain 51 was confirmed to carry the enzyme activity for methylation of Hpy51-I-recognised sequence. Hpy51-I digested genomic DNAs of enteric bacteria to less than I kb while it could not cut the genomic DNAs of H. pylori isolates. In this study, the type II restriction enzyme (Hpy51-I) of H. pylori was identified and characterized its biochemical properties, demonstrating that Hpy51-I might be one of the barriers for preventing the introduction of foreign DNAs into H. pylori.