Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation
| Autor: | Andrew M. Scharenberg, Michael T. Certo, Barry L. Stoddard, Ryo Takeuchi, Mark G. Caprara |
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| Rok vydání: | 2008 |
| Předmět: |
Models
Molecular RNA Splicing Molecular Sequence Data Sequence alignment Cleavage (embryo) Homing endonuclease Evolution Molecular 03 medical and health sciences Endonuclease chemistry.chemical_compound 0302 clinical medicine Genetics Point Mutation Amino Acid Sequence 030304 developmental biology Recombination Genetic 0303 health sciences Crystallography Base Sequence biology Nucleic Acid Enzymes Protein Stability RNA-Directed DNA Polymerase Point mutation Temperature Endonucleases Amino Acid Substitution chemistry Biochemistry RNA splicing biology.protein Directed Molecular Evolution Sequence Alignment 030217 neurology & neurosurgery DNA |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkn1007 |
| Popis: | The LAGLIDADG homing endonuclease (LHE) I-AniI has adopted an extremely efficient secondary RNA splicing activity that is beneficial to its host, balanced against inefficient DNA cleavage. A selection experiment identified point mutations in the enzyme that act synergistically to improve endonuclease activity. The amino-acid substitutions increase target affinity, alter the thermal cleavage profile and significantly increase targeted recombination in transfected cells. The RNA splicing activity is not affected by these mutations. The improvement in DNA cleavage activity is largely focused on one of the enzyme's two active sites, corresponding to a rearrangement of a lysine residue hypothesized to act as a general base. Most of the constructs isolated in the screen contain one or more mutations that revert an amino-acid identity to a residue found in one or more close homologues of I-AniI. This implies that mutations that have previously reduced the endonuclease activity of I-AniI are identified and reversed, sometimes in combination with additional 'artificial' mutations, to optimize its in vivo activity. |
| Databáze: | OpenAIRE |
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