Changing endonuclease EcoRII Tyr308 to Phe abolishes cleavage but not recognition: possible homology with the Int-family of recombinases.
Endonuclease EcoRII is one of a group of type II restriction enzymes, including Nael, Narl, BspMI, HpaII, and SacII, that require binding of an enhancer sequence to cleave DNA. Comparison of the EcoRII amino-acid sequence with the amino-acid consensus motifs that differentiate between recombinase families uncovered similarity between a 29 amino-acid sequence in the carboxyl end of EcoRII and the motif defining the integrase family of recombinases. This similarity implied that EcoRII tyrosine 308 should be involved in catalyzing hydrolysis of the scissile bond. Site-directed mutagenesis was used to mutate Tyr308 to Phe. The phenylalanine-substituted enzyme could not cleave T5 DNA under conditions in which wild-type enzyme completely cleaved this DNA. The Tyr308 to Phe mutation abolished cleavage activity but not specific binding to DNA. No evidence was found for the existence during the cleavage reaction of a covalent linkage between Tyr308 and DNA.
Topal
M D
MD
Lineberger Comprehensive Cancer Center, Department of Pathology and Curriculum in Genetics, University of North Carolina Medical School, Chapel Hill 27599-7295.
Conrad
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M
D000595:Amino Acid Sequence; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism*; D019426:Integrases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D045522:Recombinases; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014443:Tyrosine
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D045522:Recombinases; D014443:Tyrosine; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1; D004262:DNA Restriction Enzymes; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific