TY - JOUR
T1 - Redesign of the monomer-monomer interface of Cre recombinase yields an obligate heterotetrameric complex
AU - Zhang, Chi
AU - Myers, Connie A.
AU - Qi, Zongtai
AU - Mitra, Robi D.
AU - Corbo, Joseph C.
AU - Havranek, James J.
N1 - Publisher Copyright:
© The Author(s) 2015.
PY - 2015/10/15
Y1 - 2015/10/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84984685505&partnerID=8YFLogxK
U2 - 10.1093/nar/gkv901
DO - 10.1093/nar/gkv901
M3 - Article
C2 - 26365240
AN - SCOPUS:84984685505
SN - 0305-1048
VL - 43
SP - 9076
EP - 9085
JO - Nucleic acids research
JF - Nucleic acids research
IS - 18
ER -