2026-06-14T03:38:19Zhttps://repisalud.isciii.es/rest/oai/request

oai:repisalud.isciii.es:20.500.12105/66142024-11-29T17:13:50Zcom_20.500.12105_2173com_20.500.12105_2051col_20.500.12105_19597

00925njm 22002777a 4500 dc Prieto Lugo, Francisco Jesus author Redondo Bastante, Pilar author López-Méndez, Blanca author D'Abramo, Marco author Merino, Nekane author Blanco, Francisco J author Duchateau, Phillipe author Montoya, Guillermo author Molina, Rafael author 2018-07-06 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. Sci Rep. 2018; 8(1):10286. 10.1038/s41598-018-28599-0 2045-2322 2045-2322 Scientific reports 29980759 http://hdl.handle.net/20.500.12105/6614 HOMING ENDONUCLEASE SITE RECOGNITION NUCLEIC-ACIDS CLEAVAGE BINDING SEQUENCES SOFTWARE SYSTEM Understanding the indirect DNA read-out specificity of I-CreI Meganuclease