Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity.
Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.
Rimseliene
Renata
R
Institute of Biotechnology, Graiciuno 8, 2028 Vilnius, Lithuania.
Maneliene
Zita
Z
Lubys
Arvydas
A
Janulaitis
Arvydas
A
D020816:Amino Acid Motifs; D001665:Binding Sites; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D015183:Restriction Mapping; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship
D004269:DNA, Bacterial; C059642:DNA modification methylase Eco57I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015252:Deoxyribonucleases, Type II Site-Specific