Characterization of Cre-loxP interaction in the major groove: hint for structural distortion of mutant Cre and possible strategy for HIV-1 therapy.
Although the crystal structure of Cre recombinase complexed with DNA, named loxA, 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-loxP 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-loxP binding but Gln90 does not. However, the recombination activity still remained intact, although the binding between Cre and DNA substrate was not ensured.
Kim
S T
ST
School of Chemistry and Molecular Engineering, Seoul National University, Seoul 151-742, Korea.
Kim
G W
GW
Lee
Y S
YS
Park
J S
JS
D001483:Base Sequence; D017931:DNA Primers; D015658:HIV Infections / Q000628:therapy*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000302:isolation & purification; D006801:Humans; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D014764:Viral Proteins
D017931:DNA Primers; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases