Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases
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.
- Authors:
-
- Western Univ., London, ON, Canada. Schulich School of Medicine and Dentistry, Dept. of Biochemistry
- Univ. of Manitoba, Winnipeg, MB (Canada). Dept. of Microbiology
- Publication Date:
- Research Org.:
- Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Org.:
- Industrial Macromolecular Crystallography Association; USDOE Office of Science (SC); Natural Sciences and Engineering Research Council of Canada (NSERC); Canadian Institutes of Health Research (CIHR); University of Western Ontario
- OSTI Identifier:
- 1544824
- Grant/Contract Number:
- AC02-06CH11357; RPGIN-2015-04800; RGPIN-2015-03878; RGPIN-2015- 06658; MOP-89903
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nucleic Acids Research
- Additional Journal Information:
- Journal Volume: 46; Journal Issue: (22) ; 12, 2018; Journal ID: ISSN 0305-1048
- Publisher:
- Oxford University Press
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
McMurrough, Thomas A., Brown, Christopher M., Zhang, Kun, Hausner, Georg, Junop, Murray S., Gloor, Gregory B., and Edgell, David R. Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases. United States: N. p., 2018.
Web. doi:10.1093/nar/gky976.
McMurrough, Thomas A., Brown, Christopher M., Zhang, Kun, Hausner, Georg, Junop, Murray S., Gloor, Gregory B., & Edgell, David R. Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases. United States. https://doi.org/10.1093/nar/gky976
McMurrough, Thomas A., Brown, Christopher M., Zhang, Kun, Hausner, Georg, Junop, Murray S., Gloor, Gregory B., and Edgell, David R. Wed .
"Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases". United States. https://doi.org/10.1093/nar/gky976. https://www.osti.gov/servlets/purl/1544824.
@article{osti_1544824,
title = {Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases},
author = {McMurrough, Thomas A. and Brown, Christopher M. and Zhang, Kun and Hausner, Georg and Junop, Murray S. and Gloor, Gregory B. and Edgell, David R.},
abstractNote = {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.},
doi = {10.1093/nar/gky976},
journal = {Nucleic Acids Research},
number = (22) ; 12, 2018,
volume = 46,
place = {United States},
year = {2018},
month = {10}
}
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