Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Temesvári krt. 62, Hungary.
Nucleic Acids Res. 2010 Dec;38(22):8231-8. doi: 10.1093/nar/gkq676. Epub 2010 Aug 6.
The MvaI restriction endonuclease cuts 5'-CC↓AGG-3'/5'-CC↑TGG-3' sites as indicated by the arrows. N4-methylation of the inner cytosines (C(m4)CAGG/C(m4)CTGG) protects the site against MvaI cleavage. Here, we show that MvaI nicks the G-strand of the related sequence (CCGGG/CCCGG, BcnI site) if the inner cytosines are C5-methylated: C(m5)C↓GGG/CC(m5)CGG. At M.SssI-methylated SmaI sites, where two oppositely oriented methylated BcnI sites partially overlap, double-nicking leads to double-strand cleavage (CC(m5)C↓GGG/CC(m5)C↑GGG) generating fragments with blunt ends. The double-strand cleavage rate and the stringency of substrate site recognition is lower at the methylation-dependent site than at the canonical target site. MvaI is the first restriction endonuclease shown to possess, besides the 'normal' activity on its unmethylated recognition site, also a methylation-directed activity on a different sequence.
MvaI 限制内切酶在箭头所示的 5'-CC↓AGG-3'/5'-CC↑TGG-3' 位点切割。内部胞嘧啶(C(m4)CAGG/C(m4)CTGG)的 N4-甲基化保护该位点免受 MvaI 切割。在这里,我们表明如果内部胞嘧啶被 C5-甲基化:C(m5)C↓GGG/CC(m5)CGG,MvaI 会在相关序列(CCGGG/CCCGG,BcnI 位点)的 G 链上产生缺口。在 M.SssI 甲基化的 SmaI 位点,两个定向相反的甲基化 BcnI 位点部分重叠,双缺口导致双链切割(CC(m5)C↓GGG/CC(m5)C↑GGG)产生平头末端的片段。与经典靶位点相比,依赖于甲基化的位点的双链切割率和底物位点识别的严格性较低。MvaI 是第一个被证明除了在其未甲基化的识别位点上具有“正常”活性外,还在不同序列上具有甲基化指导活性的限制内切酶。