skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: 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:
 [1];  [1];  [1];  [2];  [1];  [1];  [1]
  1. Western Univ., London, ON, Canada. Schulich School of Medicine and Dentistry, Dept. of Biochemistry
  2. 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. https://doi.org/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}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 4 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Mutations altering the cleavage specificity of a homing endonuclease
journal, September 2002


Meganuclease targeting of PCSK9 in macaque liver leads to stable reduction in serum cholesterol
journal, July 2018

  • Wang, Lili; Smith, Jeff; Breton, Camilo
  • Nature Biotechnology, Vol. 36, Issue 8
  • DOI: 10.1038/nbt.4182

Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products
journal, October 2010


MegaTevs: single-chain dual nucleases for efficient gene disruption
journal, July 2014

  • Wolfs, Jason M.; DaSilva, Matthew; Meister, Sarah E.
  • Nucleic Acids Research, Vol. 42, Issue 13
  • DOI: 10.1093/nar/gku573

Displaying Variation in Large Datasets: Plotting a Visual Summary of Effect Sizes
journal, July 2016

  • Gloor, Gregory B.; Macklaim, Jean M.; Fernandes, Andrew D.
  • Journal of Computational and Graphical Statistics, Vol. 25, Issue 3
  • DOI: 10.1080/10618600.2015.1131161

The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive
journal, September 2013


A highly sensitive selection method for directed evolution of homing endonucleases
journal, October 2005


Evolution of sequence specificity in a restriction endonuclease by a point mutation
journal, July 2008

  • Saravanan, M.; Vasu, K.; Nagaraja, V.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 30
  • DOI: 10.1073/pnas.0804974105

Using Effect Size—or Why the P Value Is Not Enough
journal, September 2012


Homing endonuclease structure and function
journal, February 2005


Control of catalytic efficiency by a coevolving network of catalytic and noncatalytic residues
journal, May 2014

  • McMurrough, T. A.; Dickson, R. J.; Thibert, S. M. F.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 23
  • DOI: 10.1073/pnas.1322352111

Catalytic Versatility and Backups in Enzyme Active Sites: The Case of Serum Paraoxonase 1
journal, May 2012

  • Ben-David, Moshe; Elias, Mikael; Filippi, Jean-Jacques
  • Journal of Molecular Biology, Vol. 418, Issue 3-4
  • DOI: 10.1016/j.jmb.2012.02.042

PHENIX : building new software for automated crystallographic structure determination
journal, October 2002

  • Adams, Paul D.; Grosse-Kunstleve, Ralf W.; Hung, Li-Wei
  • Acta Crystallographica Section D Biological Crystallography, Vol. 58, Issue 11
  • DOI: 10.1107/S0907444902016657

Engineering of Large Numbers of Highly Specific Homing Endonucleases that Induce Recombination on Novel DNA Targets
journal, January 2006

  • Arnould, Sylvain; Chames, Patrick; Perez, Christophe
  • Journal of Molecular Biology, Vol. 355, Issue 3
  • DOI: 10.1016/j.jmb.2005.10.065

Engineered I-CreI Derivatives Cleaving Sequences from the Human XPC Gene can Induce Highly Efficient Gene Correction in Mammalian Cells
journal, August 2007

  • Arnould, Sylvain; Perez, Christophe; Cabaniols, Jean-Pierre
  • Journal of Molecular Biology, Vol. 371, Issue 1
  • DOI: 10.1016/j.jmb.2007.04.079

High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display
journal, September 2009

  • Jarjour, Jordan; West-Foyle, Hoku; Certo, Michael T.
  • Nucleic Acids Research, Vol. 37, Issue 20
  • DOI: 10.1093/nar/gkp726

Adaptation for Horizontal Transfer in a Homing Endonuclease
journal, March 2002


Engineering altered protein–DNA recognition specificity
journal, April 2018

  • Bogdanove, Adam J.; Bohm, Andrew; Miller, Jeffrey C.
  • Nucleic Acids Research, Vol. 46, Issue 10
  • DOI: 10.1093/nar/gky289

Type II restriction endonucleases—a historical perspective and more
journal, May 2014

  • Pingoud, Alfred; Wilson, Geoffrey G.; Wende, Wolfgang
  • Nucleic Acids Research, Vol. 42, Issue 12
  • DOI: 10.1093/nar/gku447

The CCP4 suite programs for protein crystallography
journal, September 1994


Single amino acid substitutions uncouple the DNA binding and strand scission activities of Fok I endonuclease.
journal, October 1993

  • Waugh, D. S.; Sauer, R. T.
  • Proceedings of the National Academy of Sciences, Vol. 90, Issue 20
  • DOI: 10.1073/pnas.90.20.9596

Recurrent invasion and extinction of a selfish gene
journal, November 1999

  • Goddard, M. R.; Burt, A.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 24
  • DOI: 10.1073/pnas.96.24.13880

Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity
journal, June 2016


Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases
journal, November 2008

  • Redondo, Pilar; Prieto, Jesús; Muñoz, Inés G.
  • Nature, Vol. 456, Issue 7218
  • DOI: 10.1038/nature07343

The EcoRI restriction endonuclease with bacteriophage λ DNA. Kinetic studies
journal, November 1980

  • Halford, S. E.; Johnson, N. P.; Grinsted, J.
  • Biochemical Journal, Vol. 191, Issue 2
  • DOI: 10.1042/bj1910581

Comparative meta-RNA-seq of the vaginal microbiota and differential expression by Lactobacillus iners in health and dysbiosis
journal, January 2013

  • Macklaim, Jean M.; Fernandes, Andrew D.; Di Bella, Julia M.
  • Microbiome, Vol. 1, Issue 1
  • DOI: 10.1186/2049-2618-1-12

DNA Distortion and Specificity in a Sequence-Specific Endonuclease
journal, October 2008

  • Babic, Andrea C.; Little, Elizabeth J.; Manohar, Veena M.
  • Journal of Molecular Biology, Vol. 383, Issue 1
  • DOI: 10.1016/j.jmb.2008.08.032

The role of DNA shape in protein–DNA recognition
journal, October 2009

  • Rohs, Remo; West, Sean M.; Sosinsky, Alona
  • Nature, Vol. 461, Issue 7268
  • DOI: 10.1038/nature08473

DNA Recognition and Cleavage by the LAGLIDADG Homing Endonuclease I-Cre I
journal, October 1998


Early Interrogation and Recognition of DNA Sequence by Indirect Readout
journal, December 2008


Engineering Variants of the I-SceI Homing Endonuclease with Strand-specific and Site-specific DNA-nicking Activity
journal, September 2008


Shining a light on enzyme promiscuity
journal, December 2017


Increasing cleavage specificity and activity of restriction endonuclease KpnI
journal, August 2013

  • Vasu, Kommireddy; Nagamalleswari, Easa; Zahran, Mai
  • Nucleic Acids Research, Vol. 41, Issue 21
  • DOI: 10.1093/nar/gkt734

Coevolution of a Homing Endonuclease and Its Host Target Sequence
journal, October 2007

  • Scalley-Kim, Michelle; McConnell-Smith, Audrey; Stoddard, Barry L.
  • Journal of Molecular Biology, Vol. 372, Issue 5
  • DOI: 10.1016/j.jmb.2007.07.052

LAHEDES: the LAGLIDADG homing endonuclease database and engineering server
journal, May 2012

  • Taylor, G. K.; Petrucci, L. H.; Lambert, A. R.
  • Nucleic Acids Research, Vol. 40, Issue W1
  • DOI: 10.1093/nar/gks365

Non-specific protein–DNA interactions control I-CreI target binding and cleavage
journal, April 2012

  • Molina, Rafael; Redondo, Pilar; Stella, Stefano
  • Nucleic Acids Research, Vol. 40, Issue 14
  • DOI: 10.1093/nar/gks320

Effect size, confidence interval and statistical significance: a practical guide for biologists
journal, November 2007


Alteration of Sequence Specificity of the Type II Restriction Endonuclease HincII through an Indirect Readout Mechanism
journal, May 2006

  • Joshi, Hemant K.; Etzkorn, Christopher; Chatwell, Lorentz
  • Journal of Biological Chemistry, Vol. 281, Issue 33
  • DOI: 10.1074/jbc.M512339200

Homing endonucleases from mobile group I introns: discovery to genome engineering
journal, January 2014


Redesign of extensive protein-DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
journal, March 2014

  • Takeuchi, R.; Choi, M.; Stoddard, B. L.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 11
  • DOI: 10.1073/pnas.1321030111

Substitutions in Conserved Dodecapeptide Motifs That Uncouple the DNA Binding and DNA Cleavage Activities of PI- Sce I Endonuclease
journal, March 1995

  • Gimble, Frederick S.; Stephens, Brian W.
  • Journal of Biological Chemistry, Vol. 270, Issue 11
  • DOI: 10.1074/jbc.270.11.5849

Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects
journal, August 2016


Microbiome Datasets Are Compositional: And This Is Not Optional
journal, November 2017

  • Gloor, Gregory B.; Macklaim, Jean M.; Pawlowsky-Glahn, Vera
  • Frontiers in Microbiology, Vol. 8
  • DOI: 10.3389/fmicb.2017.02224

Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation
journal, December 2008

  • Takeuchi, Ryo; Certo, Michael; Caprara, Mark G.
  • Nucleic Acids Research, Vol. 37, Issue 3
  • DOI: 10.1093/nar/gkn1007

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template
journal, September 2015

  • Sather, Blythe D.; Romano Ibarra, Guillermo S.; Sommer, Karen
  • Science Translational Medicine, Vol. 7, Issue 307
  • DOI: 10.1126/scitranslmed.aac5530

Sequence logos: a new way to display consensus sequences
journal, January 1990

  • Schneider, Thomas D.; Stephens, R. Michael
  • Nucleic Acids Research, Vol. 18, Issue 20
  • DOI: 10.1093/nar/18.20.6097

Identification of Conserved Features of Laglidadg Homing Endonucleases
journal, June 2010

  • Grishin, Alexander; Fonfara, Ines; Alexeevski, Andrei
  • Journal of Bioinformatics and Computational Biology, Vol. 08, Issue 03
  • DOI: 10.1142/S0219720010004665

Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease
journal, July 2009

  • Grizot, Sylvestre; Smith, Julianne; Daboussi, Fayza
  • Nucleic Acids Research, Vol. 37, Issue 16
  • DOI: 10.1093/nar/gkp548

Tapping natural reservoirs of homing endonucleases for targeted gene modification
journal, July 2011

  • Takeuchi, Ryo; Lambert, Abigail R.; Mak, Amanda Nga-Sze
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 32
  • DOI: 10.1073/pnas.1107719108

Understanding the indirect DNA read-out specificity of I-CreI Meganuclease
journal, July 2018


Catalytic Metal Ion Rearrangements Underline Promiscuity and Evolvability of a Metalloenzyme
journal, March 2013

  • Ben-David, Moshe; Wieczorek, Grzegorz; Elias, Mikael
  • Journal of Molecular Biology, Vol. 425, Issue 6
  • DOI: 10.1016/j.jmb.2013.01.009

Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI
journal, June 2004


Enzyme Promiscuity: A Mechanistic and Evolutionary Perspective
journal, June 2010


ANOVA-Like Differential Expression (ALDEx) Analysis for Mixed Population RNA-Seq
journal, July 2013