SITE-DIRECTED MUTAGENESIS STUDIES WITH ECORV RESTRICTION ENDONUCLEASE TO IDENTIFY REGIONS INVOLVED IN RECOGNITION AND CATALYSIS

被引:91
作者
THIELKING, V [1 ]
SELENT, U [1 ]
KOHLER, E [1 ]
WOLFES, H [1 ]
PIEPER, U [1 ]
GEIGER, R [1 ]
URBANKE, C [1 ]
WINKLER, FK [1 ]
PINGOUD, A [1 ]
机构
[1] MED HSCH HANNOVER,ZENTRUM BIOCHEM,KONSTANTY GUTSCHOW STR 8,W-3000 HANNOVER 61,GERMANY
关键词
II DNA METHYLTRANSFERASES; MODIFICATION SYSTEM; ESCHERICHIA-COLI; RI ENDONUCLEASE; SEQUENCE MOTIFS; PROTEIN; PURIFICATION; METHYLASE; CLEAVAGE; ENZYMES;
D O I
10.1021/bi00240a011
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Guided by the X-ray structure analysis of a crystalline EcoRV-d(GGGATATCCC) complex (Winkler, in preparation), we have begun to identify functionally important amino acid residues of EcoRV. We show here that Asn70, Asp74, Ser183, Asn185, Thr186, and Asn188 are most likely involved in the binding and/or cleavage of the DNA, because their conservative substitution leads to mutants of no or strongly reduced activity. In addition, C-terminal amino acid residues of EcoRV seem to be important for its activity, since their deletion inactivates the enzyme. Following the identification of three functionally important regions, we have inspected the sequences of other restriction and modification enzymes for homologous regions. It was found that two restriction enzymes that recognize similar sequences as EcoRV (DpnII and HincII), as well as two modification enzymes (M.DpnII and, in a less apparent form, M.EcoRV), have the sequence motif -SerGlyXXXAsnIleXSer- in common, which in EcoRV contains the essential Ser183 and Asn188 residues. Furthermore, the C-terminal region, shown to be essential for EcoRV, is highly homologous to a similar region in the restriction endonuclease SmaI. On the basis of these findings we propose that these restriction enzymes and to a certain extent also some of their corresponding modification enzymes interact with DNA in a similar manner.
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页码:6416 / 6422
页数:7
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