title: Redesign of the monomer-monomer interface of Cre recombinase yields an obligate heterotetrameric complex.
abstract: Cre recombinase catalyzes the cleavage and religation of DNA at loxP sites. The enzyme is a homotetramer in its functional state, and the symmetry of the protein complex enforces a pseudo-palindromic symmetry upon the loxP sequence. The Cre-lox system is a powerful tool for many researchers. However, broader application of the system is limited by the fixed sequence preferences of Cre, which are determined by both the direct DNA contacts and the homotetrameric arrangement of the Cre monomers. As a first step toward achieving recombination at arbitrary asymmetric target sites, we have broken the symmetry of the Cre tetramer assembly. Using a combination of computational and rational protein design, we have engineered an alternative interface between Cre monomers that is functional yet incompatible with the wild-type interface. Wild-type and engineered interface halves can be mixed to create two distinct Cre mutants, neither of which are functional in isolation, but which can form an active heterotetramer when combined. When these distinct mutants possess different DNA specificities, control over complex assembly directly discourages recombination at unwanted half-site combinations, enhancing the specificity of asymmetric site recombination. The engineered Cre mutants exhibit this assembly pattern in a variety of contexts, including mammalian cells.
authors.affiliation: Program in Computational and Systems Biology, Washington University in St Louis, St Louis, MO 63110, USA.
authors.forename: Chi
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Pathology and Immunology, Washington University in St Louis, St Louis, MO 63110, USA.
authors.forename: Connie A
authors.initials: CA
authors.lastname: Myers
authors.affiliation: Program in Molecular Genetics and Genomics, Washington University in St Louis, St Louis, MO 63110, USA.
authors.forename: Zongtai
authors.initials: Z
authors.lastname: Qi
authors.affiliation: Department of Genetics, Washington University in St Louis, St Louis, MO 63110, USA.
authors.forename: Robi D
authors.initials: RD
authors.lastname: Mitra
authors.affiliation: Department of Pathology and Immunology, Washington University in St Louis, St Louis, MO 63110, USA.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Corbo
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University in St Louis, St Louis, MO 63110, USA Havranek@biochem.wustl.edu.
authors.forename: James J
authors.initials: JJ
authors.lastname: Havranek
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D019426:Integrases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D055503:Protein Multimerization; D011995:Recombination, Genetic
chemical_list: D004247:DNA; C045073:Cre recombinase; D019426:Integrases