Taylor J D, Goodall A J, Vermote C L, Halford S E
Department of Biochemistry, University of Bristol, U.K.
Biochemistry. 1990 Dec 4;29(48):10727-33. doi: 10.1021/bi00500a003.
The EcoRV restriction/modification system consists of two enzymes that recognize the DNA sequence GATATC. The EcoRV restriction endonuclease cleaves DNA at this site, but the DNA of Escherichia coli carrying the EcoRV system is protected from this reaction by the EcoRV methyltransferase. However, in vitro, the EcoRV nuclease also cleaves DNA at most sites that differ from the recognition sequence by one base pair. Though the reaction of the nuclease at these sites is much slower than that at the cognate site, it still appears to be fast enough to cleave the chromosome of the cell into many fragments. The possibility that the EcoRV methyltransferase also protects the noncognate sites on the chromosome was examined. The modification enzyme methylated alternate sites in vivo, but these were not the same as the alternate sites for the nuclease. The excess methylation was found at GATC sequences, which are also the targets for the dam methyltransferase of E. coli, a protein that is homologous to the EcoRV methyltransferase. Methylation at these sites gave virtually no protection against the EcoRV nuclease: even when the EcoRV methyltransferase had been overproduced, the cellular DNA remained sensitive to the EcoRV nuclease at its noncognate sites. The viability of E. coli carrying the EcoRV restriction/modification system was found instead to depend on the activity of DNA ligase. Ligase appears to proofread the EcoRV R/M system in vivo: DNA, cut initially in one strand at a noncognate site for the nuclease, is presumably repaired by ligase before the scission of the second strand.
EcoRV限制/修饰系统由两种识别DNA序列GATATC的酶组成。EcoRV限制性内切核酸酶在此位点切割DNA，但携带EcoRV系统的大肠杆菌DNA受到EcoRV甲基转移酶的保护而免受此反应影响。然而，在体外，EcoRV核酸酶也会在大多数与识别序列相差一个碱基对的位点切割DNA。尽管核酸酶在这些位点的反应比在同源位点慢得多，但似乎仍然足够快地将细胞染色体切割成许多片段。研究了EcoRV甲基转移酶是否也保护染色体上的非同源位点。修饰酶在体内甲基化交替位点，但这些位点与核酸酶的交替位点不同。在GATC序列上发现了过量甲基化，这些序列也是大肠杆菌dam甲基转移酶的作用靶点，dam甲基转移酶是一种与EcoRV甲基转移酶同源的蛋白质。这些位点的甲基化几乎不能保护细胞免受EcoRV核酸酶的作用：即使EcoRV甲基转移酶过量表达，细胞DNA在其非同源位点对EcoRV核酸酶仍保持敏感。相反，发现携带EcoRV限制/修饰系统的大肠杆菌的生存能力取决于DNA连接酶的活性。连接酶似乎在体内对EcoRV限制/修饰系统进行校对：最初在核酸酶的非同源位点单链切割的DNA，大概在第二条链断裂之前由连接酶修复。
