Document Detail


Engineering variants of the I-SceI homing endonuclease with strand-specific and site-specific DNA-nicking activity.
MedLine Citation:
PMID:  18644379     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Yan Niu; Kristen Tenney; Hongye Li; Frederick S Gimble
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2008-07-11
Journal Detail:
Title:  Journal of molecular biology     Volume:  382     ISSN:  1089-8638     ISO Abbreviation:  J. Mol. Biol.     Publication Date:  2008 Sep 
Date Detail:
Created Date:  2008-08-19     Completed Date:  2008-09-04     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  2985088R     Medline TA:  J Mol Biol     Country:  England    
Other Details:
Languages:  eng     Pagination:  188-202     Citation Subset:  IM    
Affiliation:
Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Base Sequence
Conserved Sequence
Crystallography, X-Ray
DNA Breaks, Single-Stranded*
DNA, Superhelical / chemistry,  genetics
Deoxyribonucleases, Type II Site-Specific / metabolism*
Kinetics
Molecular Sequence Data
Mutant Proteins / metabolism*
Nucleic Acid Conformation
Plasmids / genetics
Protein Engineering*
Protein Structure, Secondary
Saccharomyces cerevisiae Proteins
Substrate Specificity
Thermodynamics
Time Factors
Grant Support
ID/Acronym/Agency:
GM 070553/GM/NIGMS NIH HHS; R01 GM070553-01/GM/NIGMS NIH HHS; R01 GM070553-02/GM/NIGMS NIH HHS; R01 GM070553-03/GM/NIGMS NIH HHS; R01 GM070553-04/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/DNA, Superhelical; 0/Mutant Proteins; 0/Saccharomyces cerevisiae Proteins; EC 3.1.21.-/SCEI protein, S cerevisiae; EC 3.1.21.4/Deoxyribonucleases, Type II Site-Specific
Comments/Corrections

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