Redesign of the monomer–monomer interface of Cre recombinase yields an obligate heterotetrameric complex
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...
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| Format: | Online |
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Oxford University Press
2015
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605323/ |
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pubmed-46053232015-10-19 Redesign of the monomer–monomer interface of Cre recombinase yields an obligate heterotetrameric complex Zhang, Chi Myers, Connie A. Qi, Zongtai Mitra, Robi D. Corbo, Joseph C. Havranek, James J. Synthetic Biology and Bioengineering 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. Oxford University Press 2015-10-15 2015-10-10 /pmc/articles/PMC4605323/ /pubmed/26365240 http://dx.doi.org/10.1093/nar/gkv901 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Zhang, Chi Myers, Connie A. Qi, Zongtai Mitra, Robi D. Corbo, Joseph C. Havranek, James J. |
| spellingShingle |
Zhang, Chi Myers, Connie A. Qi, Zongtai Mitra, Robi D. Corbo, Joseph C. Havranek, James J. Redesign of the monomer–monomer interface of Cre recombinase yields an obligate heterotetrameric complex |
| author_facet |
Zhang, Chi Myers, Connie A. Qi, Zongtai Mitra, Robi D. Corbo, Joseph C. Havranek, James J. |
| author_sort |
Zhang, Chi |
| title |
Redesign of the monomer–monomer interface of Cre recombinase yields an obligate heterotetrameric complex |
| title_short |
Redesign of the monomer–monomer interface of Cre recombinase yields an obligate heterotetrameric complex |
| title_full |
Redesign of the monomer–monomer interface of Cre recombinase yields an obligate heterotetrameric complex |
| title_fullStr |
Redesign of the monomer–monomer interface of Cre recombinase yields an obligate heterotetrameric complex |
| title_full_unstemmed |
Redesign of the monomer–monomer interface of Cre recombinase yields an obligate heterotetrameric complex |
| title_sort |
redesign of the monomer–monomer interface of cre recombinase yields an obligate heterotetrameric complex |
| description |
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. |
| publisher |
Oxford University Press |
| publishDate |
2015 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605323/ |
| _version_ |
1613488025869746176 |