Engineering Variants of the I-SceI Homing Endonuclease with Strand-specific and Site-specific DNA-nicking Activity
| Autor: | Kristen Tenney, Hongye Li, Frederick S. Gimble, Yan Niu |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Time Factors Stereochemistry Molecular Sequence Data Crystallography X-Ray Protein Engineering Cleavage (embryo) Protein Structure Secondary Article Homing endonuclease Substrate Specificity chemistry.chemical_compound Plasmid Structural Biology Amino Acid Sequence DNA Breaks Single-Stranded Enzyme kinetics Deoxyribonucleases Type II Site-Specific Molecular Biology Conserved Sequence Base Sequence biology DNA Superhelical Nicking enzyme Protein engineering Kinetics chemistry Biochemistry Phosphodiester bond biology.protein Nucleic Acid Conformation Thermodynamics Mutant Proteins DNA Plasmids |
| Zdroj: | Journal of Molecular Biology. 382:188-202 |
| ISSN: | 0022-2836 |
| DOI: | 10.1016/j.jmb.2008.07.010 |
| Popis: | 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 four base-pairs in length. 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 sequentially cleaves the two DNA strands. 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, Lys-122 and Lys-223, markedly reduces top or 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. |
| Databáze: | OpenAIRE |
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