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Characterization of Cre– lox P interaction in the major groove: Hint for structural distortion of mutant Cre and possible strategy for HIV‐1 therapy
Author(s) -
Kim Sungtae,
Kim Gyoungwon,
Lee Youngsam,
Park Jongsang
Publication year - 2000
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/1097-4644(20010301)80:3<321::aid-jcb40>3.0.co;2-c
Subject(s) - mutant , cre recombinase , dna , biology , mutagenesis , mutation , chemistry , microbiology and biotechnology , biochemistry , genetics , gene , transgene , genetically modified mouse
Abstract Although the crystal structure of Cre recombinase complexed with DNA, named lox A, was elucidated a couple of years ago, it has not yet been determined which amino acids of the protein are involved in the specific Cre– lox P interaction. Arg259 and Gln90 interact with DNA substrate in the major groove from which the specificity of protein–DNA interaction comes. In this study, we substituted these residues for other amino acids. Also, two mutated DNA substrates were constructed. In each mutant, one of the bases that interact with Arg259 or Gln90 was changed into another base. In vitro binding assays and recombination assays of variant lox sites with wild‐type and mutant‐type Cre revealed that Arg259 plays a key role in Cre– lox P binding but Gln90 does not. However, the recombination activity still remained intact, although the binding between Cre and DNA substrate was not ensured. J. Cell. Biochem. 80:321–327, 2001. © 2001 Wiley‐Liss, Inc.