Som S, Friedman S
Department of Pharmacology, State University of New York Health Science Center, Brooklyn 11203.
J Biol Chem. 1994 Oct 21;269(42):25986-91.
DNA(cytosine-5)-methylases form tight complexes at their methylation sites when the target cytosine residue is substituted by analogs such as 5-azacytosine or 5-fluorocytosine. To test whether such complexes can block RNA transcription in vitro, template DNA-containing methylation sites were prepared, in which cytosine residues in either the (+)- or (-)-strand were substituted by the analogs. Such templates, irrespective of the strand in which substitution was made, could effectively block the elongation of RNA at specific sites when complexed with EcoRII methylase. The protein-DNA complex probably prevents the unwinding of the template strands or might directly present itself as a steric block to the advancing RNA polymerase. RNA synthesis was also inhibited at specific sites due to complex formation between azacytosine-containing DNA and two other methylases, HhaI and HpaII. The 3' ends of the interrupted transcripts were mapped and were found to lie within 13-14, 13, and 23 nucleotides of the binding sites on the (-)-strand for HhaI, HpaII, and EcoRII methylases, respectively. Exonuclease III footprinting revealed that the boundaries of the complexed methylases, HhaI, HpaII, and EcoRII, on the (-)-strand were within 2-3, 1-2, and 9-10 nucleotides, respectively, of the last nucleotide copied by the RNA polymerase.
当靶胞嘧啶残基被诸如5-氮杂胞嘧啶或5-氟胞嘧啶等类似物取代时，DNA（胞嘧啶-5）-甲基化酶在其甲基化位点形成紧密复合物。为了测试此类复合物是否能在体外阻断RNA转录，制备了含有甲基化位点的模板DNA，其中正义链或反义链中的胞嘧啶残基被类似物取代。此类模板，无论取代发生在哪一条链上，与EcoRII甲基化酶复合时都能在特定位点有效阻断RNA的延伸。蛋白质-DNA复合物可能会阻止模板链解旋，或者可能直接作为空间位阻阻碍前进的RNA聚合酶。由于含氮杂胞嘧啶的DNA与另外两种甲基化酶HhaI和HpaII之间形成复合物，RNA合成在特定位点也受到抑制。对中断转录本的3'末端进行定位，发现它们分别位于HhaI、HpaII和EcoRII甲基化酶在反义链上结合位点的13 - 14、13和23个核苷酸范围内。核酸外切酶III足迹分析表明，反义链上与HhaI、HpaII和EcoRII复合的甲基化酶的边界分别在RNA聚合酶复制的最后一个核苷酸的2 - 3、1 - 2和9 - 10个核苷酸范围内。
