Non-specific protein-DNA interactions control I-CreI target binding and cleavage

Rafael Molina; Pilar Redondo; Stefano Stella; Marco Marenchino; Marco D'Abramo; Francesco Luigi Gervasio; Jean Charles Epinat; Julien Valton; Silvestre Grizot; Phillipe Duchateau; Jesús Prieto; Guillermo Montoya

doi:10.1093/nar/gks320

Non-specific protein-DNA interactions control I-CreI target binding and cleavage

Rafael Molina, Pilar Redondo, Stefano Stella, Marco Marenchino, Marco D'Abramo, Francesco Luigi Gervasio, Jean Charles Epinat, Julien Valton, Silvestre Grizot, Phillipe Duchateau, Jesús Prieto, Guillermo Montoya^*

^*Corresponding author for this work

Protein Structure and Function Program

20 Citations (Scopus)

Abstract

Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications.

Original language	English
Journal	Nucleic Acids Research
Volume	40
Issue number	14
Pages (from-to)	6936-6945
Number of pages	10
ISSN	0305-1048
DOIs	https://doi.org/10.1093/nar/gks320
Publication status	Published - 1 Aug 2012

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title = "Non-specific protein-DNA interactions control I-CreI target binding and cleavage",

abstract = "Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications.",

author = "Rafael Molina and Pilar Redondo and Stefano Stella and Marco Marenchino and Marco D'Abramo and Gervasio, {Francesco Luigi} and {Charles Epinat}, Jean and Julien Valton and Silvestre Grizot and Phillipe Duchateau and Jes{\'u}s Prieto and Guillermo Montoya",

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AU - Molina, Rafael

AU - Redondo, Pilar

AU - Stella, Stefano

AU - Marenchino, Marco

AU - D'Abramo, Marco

AU - Gervasio, Francesco Luigi

AU - Charles Epinat, Jean

AU - Valton, Julien

AU - Grizot, Silvestre

AU - Duchateau, Phillipe

AU - Prieto, Jesús

AU - Montoya, Guillermo

PY - 2012/8/1

Y1 - 2012/8/1

N2 - Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications.

AB - Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications.

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Non-specific protein-DNA interactions control I-CreI target binding and cleavage

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