Understanding the indirect DNA read-out specificity of I-CreI Meganuclease

Jesús Prieto,Nekane Merino,Marco D’Abramo,Rafael Molina,Guillermo Montoya,Phillipe Duchateau,Francisco J Blanco,Blanca López-Méndez,Pilar Redondo

doi:10.1038/s41598-018-28599-0

Abstract

The high DNA specificity of homing endonucleases makes them a powerful protein scaffold to engineer enzymes for genome manipulation. Understanding their molecular recognition of DNA is an important prerequisite to generate engineered enzymes able to cleave DNA in specific desired genome sites. Protein-DNA recognition studies have been mostly focused on specific direct contacts between amino acid side chains and bases to redesign the binding interface. However, the important role of indirect readout in the central region of the target DNA of the homing endonuclease I-CreI suggested that indirect readout may play a key role in the redesign of protein-DNA interactions. The sequences of the I-CreI central substrate region, 2NN, along with the adjacent 5NNN, are key for substrate cleavage. Here, we analyse the mechanism of target discrimination at the 5NNN region by the I-CreI protein, revealing its critical role in the location and occupancy of the catalytic metal ions, which is crucial for cleavage. Our data highlight the importance of indirect readout for target DNA cleavage, thus aiding I-CreI engineering when targeting new DNA sequences.

Highlights

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Previous studies have pointed out that I-CreI meganuclease has a preference for certain nucleotides at the 5NNN of its target sequence, and that a G in position −4 strongly hampers cleavage[6,9]
The redesign of the protein-DNA binding properties in this scaffold has been performed taking into account the specific protein-DNA contacts. In this sense, understanding of the 2NN region role in target recognition and cleavage had a strong impact in meganuclease engineering targeting new DNA sequences that avoid the presence of the non-preferred bases in the central region, optimizing meganuclease tailoring

Summary

Introduction

8 rue de la Croix Jarry, 75013, Paris, France. Correspondence and requests for materials should be addressed to R.M. The influence of the central sequence was explained by its topology, showing a mechanism governing target discrimination not based on specific protein–DNA contacts[12]. The 2NN region affects the active site rearrangement, the proper protein-DNA complex binding and catalytic ion positioning to lead the cleavage. The results suggest a mechanism controlling target discrimination based on specific protein-DNA contacts, but on the proper positioning of the catalytic ion. These findings, in line with previous reports[12] allow us to further rationalize the search for new target sequences in the development of new-engineered homing endonucleases for therapeutic and biotechnological applications

Methods

Results

Conclusion