MG2+ CONFERS DNA-BINDING SPECIFICITY TO THE ECORV RESTRICTION ENDONUCLEASE

被引:81
作者
THIELKING, V [1 ]
SELENT, U [1 ]
KOHLER, E [1 ]
LANDGRAF, A [1 ]
WOLFES, H [1 ]
ALVES, J [1 ]
PINGOUD, A [1 ]
机构
[1] MED HSCH HANNOVER, ZENTRUM BIOCHEM, KONSTANTY GUTSCHOW STR 8, W-3000 HANNOVER 61, GERMANY
关键词
D O I
10.1021/bi00130a001
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
The EcoRV mutant D90A which carries an amino acid substitution in its active center does not cleave DNA. Therefore, it is possible to perform DNA binding experiments with the EcoRV-D90A mutant both in the absence and in the presence of Mg2+. Like wild-type EcoRV [Taylor et al. (1991) Biochemistry 30, 8743-8753], it does not show a pronounced specificity for binding to its recognition site in the absence of Mg2+ as judged by the appearance of multiple shifted bands in an electrophoretic mobility shift assay with a 377-bp DNA fragment carrying a single EcoRV recognition sequence. In the presence of Mg2+, however, only one band corresponding to a 1:1 complex appears even with a high excess of protein over DNA. This complex most likely is the specific one, because its formation is suppressed much more effectively by a 13-bp oligodeoxynucleotide with an EcoRV site than by a corresponding oligodeoxynucleotide without an EcoRV site. The preferential interaction of the EcoRV-D90A mutant with specific DNA in the presence of Mg2+ was also demonstrated directly: a 20-bp oligodeoxynucleotide with an EcoRV site is bound with K(Ass) = 4 X 10(8) M-1, while a corresponding oligodeoxynucleotide without an EcoRV site is bound with K(Ass) less-than-or-equal-to 1 X 10(5) M-1. From these data it appears that Mg2+ confers DNA binding specificity to this mutant by lowering the affinity to nonspecific sites and raising the affinity to specific sites as compared to binding in the absence of Mg2+. It is concluded that this is also true for wild-type EcoRV.
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页码:3727 / 3732
页数:6
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