Asn141 is essential for DNA recognition by EcoRI restriction endonuclease

FEBS Letters

Fritz, Andreas²  Küster, Wolfgang¹  Alves, Jürgen¹ 

[1] Zentrum Biochemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany;Institut für Pathologie, GSG-Forschungszentrum für Umwelt und Gesundheit GmbH, Ingolstädter Landstr. 1, D-85758 Oberschleißheim, Germany

关键词: DNA-protein interaction;    Type II restriction enzyme;    Sequence specificity;    Site-directed mutagenesis;    Restriction modification;    Hydrogen bond;    CD;    circular dichroism;   

DOI  :  10.1016/S0014-5793(98)01274-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The amino acid residue Asn¹⁴¹ of the restriction endonuclease EcoRI was proposed to make three hydrogen bonds to both adenine residues within the recognition sequence -GAATTC-. We have mutated Asn¹⁴¹ to alanine, aspartate, serine, and tyrosine. Only the serine mutant is active under normal buffer conditions although 1000-fold less than wild-type EcoRI. The alanine and aspartate mutants can be activated by Mn²⁺. At acidic pH the latter mutant becomes even more active than the wild-type enzyme in the presence of Mn²⁺. We conclude that Asn¹⁴¹ is essential for DNA recognition and that serine can partly substitute it.

【 授权许可】

Unknown