ArticleJournal of molecular biology2008
Engineering variants of the I-SceI homing endonuclease with strand-specific and site-specific DNA-nicking activity.
Article in Journal of molecular biology, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 41 papers.
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Who cites it
41 citing papers in PubMed, 69 citations in OpenAlex.
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- Cryo-EM structures of DNA-free and DNA-bound BsaXI: architecture of a Type IIB restriction-modification enzyme.Nucleic acids research · 2025Article
- Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases.Nature biotechnology · 2023Article
- Essential Role for an Isoform of Escherichia coli Translation Initiation Factor IF2 in Repair of Two-Ended DNA Double-Strand Breaks.Journal of bacteriology · 2022Article
- NgAgo possesses guided DNA nicking activity.Nucleic acids research · 2021Article
- Genome Editing in Bacteria: CRISPR-Cas and Beyond.Microorganisms · 2021Review
- Protocol: A Multiplexed Reporter Assay to Study Effects of Chromatin Context on DNA Double-Strand Break Repair.Frontiers in genetics · 2021Article
- A robust CRISPR-Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair.Nucleic acids research · 2020Article
- Synthetic control of plasmid replication enables target- and self-curing of vectors and expedites genome engineering ofMetabolic engineering communications · 2020Article
- Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants.Rice (New York, N.Y.) · 2019Review
- Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases.Nucleic acids research · 2018Article
- Initiation of homologous recombination at DNA nicks.Nucleic acids research · 2018Review
- Assaying Repair at DNA Nicks.Methods in enzymology · 2018Article
- A double-strand break can trigger immunoglobulin gene conversion.Nucleic acids research · 2017Article
- The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.DNA repair · 2016Review
- The Structural Basis of Asymmetry in DNA Binding and Cleavage as Exhibited by the I-SmaMI LAGLIDADG Meganuclease.Journal of molecular biology · 2016Article
- Engineering a Nickase on the Homing Endonuclease I-DmoI Scaffold.The Journal of biological chemistry · 2015Article
- Aptamer-guided gene targeting in yeast and human cells.Nucleic acids research · 2014Article
- To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.PloS one · 2014Article
- Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice.Nucleic acids research · 2014Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
The number of strand-specific nicking endonucleases that are currently available for laboratory procedures and applications in vivo is limited, and none is sufficiently specific to nick single target sites within complex genomes. The extreme target specificity of homing endonucleases makes them attractive candidates for engineering high-specificity nicking endonucleases. I-SceI is a monomeric homing enzyme that recognizes an 18 bp asymmetric target sequence, and cleaves both DNA strands to leave 3'-overhangs of 4 bp. In single turnover experiments using plasmid substrates, I-SceI generates transient open circle intermediates during the conversion of supercoiled to linear DNA, indicating that the enzyme cleaves the two DNA strands sequentially. A novel hairpin substrate was used to demonstrate that although wild-type I-SceI cleaves either the top or bottom DNA strand first to generate two nicked DNA intermediates, the enzyme has a preference for cleaving the bottom strand. The kinetics data are consistent with a parallel sequential reaction mechanism. Substitution of two pseudo-symmetric residues, Lys122 and Lys223, markedly reduces top and bottom-strand cleavage, respectively, to generate enzymes with significant strand- and sequence-specific nicking activity. The two active sites are partially interdependent, since alterations to one site affect the second. The kinetics analysis is consistent with X-ray crystal structures of I-SceI/DNA complexes that reveal a role for the lysines in establishing important solvent networks that include nucleophilic water molecules thought to attack the scissile phosphodiester bonds.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.