Cre mutants with altered DNA binding properties.
The recombinase Cre of bacteriophage P1 is a member of the family of site-specific recombinases and integrases that catalyze inter- and intramolecular DNA rearrangements. To understand how this protein specifically recognizes its target sequence, we constructed Cre mutants with amino acid substitutions in different positions of the presumptive DNA binding region. Here we present the results of in vitro DNA binding and in vivo recombination experiments with these Cre mutants. Most substitutions of presumptive DNA-binding amino acids in in vitro tests resulted either in the loss of target binding or in a broadening of target recognition specificity. Of the mutations resulting in a broadening of target specificity, one, N317A, results in a reduced recombination efficiency with the wild-type loxP target but recombines, in contrast to wild-type Cre, in in vivo experiments, with a symmetric variant of the wild-type target sequence. This target variant differs from wild-type loxP by the symmetric C to A replacement in position 6 of the inverted repeats. We propose a common multihelical DNA binding motif for the family of integrases and recombinases. This model implies a major structural rearrangement for the DNA binding region of lambda integrase, analogous to the structural rearrangements of the DNA binding motifs of other proteins when contacting their target DNA.
Institute for Genetics, University of Cologne, Weyertal 121, D-50931 Cologne, Germany.
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Hartung
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Kisters-Woike
D017101:Bacteriophage P1 / Q000201:enzymology; D001665:Binding Sites / Q000235:genetics; D002474:Cell-Free System; D003434:Crossing Over, Genetic; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011995:Recombination, Genetic; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases