The Ecl18kI restriction-modification system:: cloning, expression, properties of the purified enzymes

被引:11
作者
Denjmukhametov, MM
Brevnov, MG
Zakharova, MV
Repyk, AV
Solonin, AS
Petrauskene, OV
Gromova, ES [1 ]
机构
[1] Moscow MV Lomonosov State Univ, Dept Chem, Moscow 119899, Russia
[2] Russian Acad Sci, Inst Biochem & Physiol Microorganisms, Pushchino 142292, Moscow Region, Russia
[3] Moscow MV Lomonosov State Univ, Belozersky Inst Physicochem Biol, Moscow 119899, Russia
基金
俄罗斯基础研究基金会;
关键词
Ecl18kI DNA methyltransferase; Ecl18kI restriction endonuclease; purification; abasic substrate analog;
D O I
10.1016/S0014-5793(98)00921-1
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Ecl18kI is a type II restriction-modification system isolated from Enterobacter cloaceae 18kI strain. Genes encoding Ecl18kI methyltransferase (M.Ecl18kI) and Ecl18kI restriction endonuclease (R.Ecl18kI) have been cloned and expressed in Escherichia coli. These enzymes recognize the 5'...down arrow CCNGG...3' sequence in DNA; M.Ecl18kI methylates the C5 carbon atom of the inner dC residue and R.Ecl18kI cuts DNA as shown by the arrow, The restriction endonuclease and the methyltransferase were purified from E. coli B834 [p18Ap1] cells to near homogeneity, The restriction endonuclease is present in the solution as a tetramer, while the methyltransferase is a monomer, The interactions of M.Ecl18kI and R.Ecl18kI with 1,2-dideoxy-D-ribofuranose containing DNA duplexes were investigated. The target base flipping-out mechanism is applicable in the case of M.Ecl18kI. Correct cleavage of the abasic substrates by R.Ecl18kI is accompanied by non-canonical hydrolysis of the modified strand. (C) 1998 Federation of European Biochemical Societies.
引用
收藏
页码:233 / 236
页数:4
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