Ecl18kI 限制修饰系统：克隆、表达及纯化酶的特性

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

作者信息

Denjmukhametov M M, Brevnov M G, Zakharova M V, Repyk A V, Solonin A S, Petrauskene O V, Gromova E S

机构信息

Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region.

出版信息

FEBS Lett. 1998 Aug 21;433(3):233-6. doi: 10.1016/s0014-5793(98)00921-1.

DOI:10.1016/s0014-5793(98)00921-1

PMID:9744801

Abstract

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'.../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.

摘要

Ecl18kI是一种从阴沟肠杆菌18kI菌株中分离出来的II型限制修饰系统。编码Ecl18kI甲基转移酶（M.Ecl18kI）和Ecl18kI限制性内切酶（R.Ecl18kI）的基因已在大肠杆菌中克隆并表达。这些酶识别DNA中的5'.../CCNGG...3'序列；M.Ecl18kI使内部dC残基的C5碳原子甲基化，R.Ecl18kI按箭头所示切割DNA。限制性内切酶和甲基转移酶从大肠杆菌B834 [p18Ap1]细胞中纯化至接近均一。限制性内切酶以四聚体形式存在于溶液中，而甲基转移酶是单体。研究了M.Ecl18kI和R.Ecl18kI与含1,2-二脱氧-D-核糖呋喃糖的DNA双链体的相互作用。目标碱基翻转机制适用于M.Ecl18kI的情况。R.Ecl18kI对无碱基底物的正确切割伴随着修饰链的非经典水解。