A study of the Asp110-Glu112 region of EcoRII restriction endonuclease by site-directed mutagenesis.
Site-directed mutagenesis of the ecoRII gene has been used to search for the active site of the EcoRII restriction endonuclease. Plasmids with point mutations in ecoRII gene resulting in substitutions of amino acid residues in the Asp110-Glu112 region of the EcoRII endonuclease (Asp110 --> Lys, Asn, Thr, Val, or Ile; Pro111 --> Arg, His, Ala, or Leu; Glu112 --> Lys, Gln, or Asp) have been constructed. When expressed in E. coli, all these plasmids displayed EcoRII endonuclease activity. We also constructed a plasmid containing a mutant ecoRII gene with deletion of the sequence coding the Gln109-Pro111 region of the protein. This mutant protein had no EcoRII endonuclease activity. The data suggest that Asp110, Pro111, and Glu112 residues do not participate in the formation of the EcoRII active site. However, this region seems to be relevant for the formation of the tertiary structure of the EcoRII endonuclease.
Sergeev
V N
VN
School of Chemistry and Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899, Russia.
Chalov
S E
SE
Drutsa
V L
VL
Gromova
E S
ES
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D017931:DNA Primers; D005973:Glutamine; D001224:Aspartic Acid; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific