Molecular analysis of restriction endonuclease EcoRII from Escherichia coli reveals precise regulation of its enzymatic activity by autoinhibition.
Bacterial restriction endonuclease EcoRII requires two recognition sites to cleave DNA. Proteolysis of EcoRII revealed the existence of two stable domains, EcoRII-N and EcoRII-C. Reduction of the enzyme to its C-terminal domain, EcoRII-C, unleashed the enzyme activity; this truncated form no longer needed two recognition sites and cleaved DNA much more efficiently than EcoRII wild-type. The crystal structure of EcoRII showed that probably the N-terminal domain sterically occludes the catalytic site, thus apparently controlling the cleavage activity. Based on these data, EcoRII was the first restriction endonuclease for which an autoinhibition mechanism as regulatory strategy was proposed. In this study, we probed this assumption and searched for the inhibitory element that mediates autoinhibition. Here we show that repression of EcoRII-C is achieved by addition of the inhibitory domain EcoRII-N or by single soluble peptides thereof in trans. Moreover, we perturbed contacts between the N- and the C-terminal domain of EcoRII by site-directed mutagenesis and proved that beta-strand B1 and alpha-helix H2 are essential for autoinhibition; deletion of either secondary structural element completely relieved EcoRII autoinhibition. This potent regulation principle that keeps EcoRII enzyme activity controlled might protect bacteria against suicidal restriction of rare unmodified recognition sites in the cellular genome.
Szczepek
Michal
M
Institute of Medical Virology, Helmut-Ruska-Haus, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
Mackeldanz
Petra
P
Möncke-Buchner
Elisabeth
E
Alves
Jürgen
J
Krüger
Detlev H
DH
Reuter
Monika
M
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D004247:DNA; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific