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- W2895918744 abstract "LAGLIDADG homing endonucleases (meganucleases) are site-specific mobile endonucleases that can be adapted for genome-editing applications. However, one problem when reprogramming meganucleases on non-native substrates is indirect readout of DNA shape and flexibility at the central 4 bases where cleavage occurs. To understand how the meganuclease active site regulates DNA cleavage, we used functional selections and deep sequencing to profile the fitness landscape of 1600 I-LtrI and I-OnuI active site variants individually challenged with 67 substrates with central 4 base substitutions. The wild-type active site was not optimal for cleavage on many substrates, including the native I-LtrI and I-OnuI targets. Novel combinations of active site residues not observed in known meganucleases supported activity on substrates poorly cleaved by the wild-type enzymes. Strikingly, combinations of E or D substitutions in the two metal-binding residues greatly influenced cleavage activity, and E184D variants had a broadened cleavage profile. Analyses of I-LtrI E184D and the wild-type proteins co-crystallized with the non-cognate AACC central 4 sequence revealed structural differences that correlated with kinetic constants for cleavage of individual DNA strands. Optimizing meganuclease active sites to enhance cleavage of non-native central 4 target sites is a straightforward addition to engineering workflows that will expand genome-editing applications." @default.
- W2895918744 created "2018-10-26" @default.
- W2895918744 creator A5002669105 @default.
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- W2895918744 creator A5038673996 @default.
- W2895918744 creator A5049609503 @default.
- W2895918744 creator A5052179215 @default.
- W2895918744 date "2018-12-14" @default.
- W2895918744 modified "2023-09-29" @default.
- W2895918744 title "Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases" @default.
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- W2895918744 doi "https://doi.org/10.2210/pdb6bcf/pdb" @default.
- W2895918744 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6294521" @default.
- W2895918744 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30357419" @default.
- W2895918744 hasPublicationYear "2018" @default.
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