Kautiainen T L, Jones P A
Biochemistry. 1985 Sep 24;24(20):5575-81. doi: 10.1021/bi00341a043.
A novel system to study the methylation of newly synthesized DNA in isolated nuclei was developed. Approximately 2.5% of cytosine residues incorporated into nascent DNA became methylated by endogenous methylase(s), and the level of DNA modification was reduced by methylation inhibitors. DNA synthesis and methylation were dependent on separate cytosol factors. The cytosol factor or factors required for DNA methylation were sensitive to trypsin digestion and were precipitable by (NH4)2SO4, suggesting that they were proteinaceous. Time-course experiments revealed a short lag of approximately 20 s between synthesis and methylation in nuclei. The DNAs produced in these nuclei were a mixed population of low molecular weight fragments and higher molecular weight fragments shown to be short extension of existing replicons. The methylation level found in low molecular weight DNA was lower than that found in bulk L1210 DNA, indicating that further methylation events might take place after ligation of small fragments. These data suggest that newly synthesized DNA is a good substrate for methylase enzymes and that nuclear cytoplasmic interactions may be important in controlling inheritance of methylation patterns.
开发了一种用于研究分离细胞核中新合成DNA甲基化的新系统。掺入新生DNA中的胞嘧啶残基约2.5%被内源性甲基化酶甲基化，并且DNA修饰水平因甲基化抑制剂而降低。DNA合成和甲基化依赖于不同的胞质溶胶因子。DNA甲基化所需的一种或多种胞质溶胶因子对胰蛋白酶消化敏感，并且可被硫酸铵沉淀，这表明它们是蛋白质性质的。时间进程实验显示细胞核中合成与甲基化之间存在约20秒的短暂延迟。这些细胞核中产生的DNA是低分子量片段和高分子量片段的混合群体，高分子量片段显示为现有复制子的短延伸。在低分子量DNA中发现的甲基化水平低于在大量L1210 DNA中发现的水平，这表明小片段连接后可能会发生进一步的甲基化事件。这些数据表明新合成的DNA是甲基化酶的良好底物，并且核质相互作用在控制甲基化模式的遗传中可能很重要。
