Engineering variants of the I-SceI homing endonuclease with strand-specific and site-specific DNA-nicking activity.
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.
Niu
Yan
Y
Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.
Tenney
Kristen
K
Li
Hongye
H
Gimble
Frederick S
FS
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D053904:DNA Breaks, Single-Stranded; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013816:Thermodynamics; D013997:Time Factors
D004278:DNA, Superhelical; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific