Concurrent replication and methylation at mammalian origins of replication
Concurrent replication and methylation at mammalian origins of replication
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DOI：
10.1128/mcb.18.6.3475
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发表时间：
1998-06-01
影响因子：
5.3
通讯作者：
Zannis-Hadjopoulos, M
中科院分区：
文献类型：
--
作者：
Araujo, FD;Knox, JD;Zannis-Hadjopoulos, M
对大肠杆菌的观察表明，复制和甲基化之间的滞后调节了复制的起始。为了解决在哺乳动物细胞中是否存在类似机制的问题，我们已经确定了哺乳动物细胞中复制起始和甲基化之间的时间关系，包括全面水平和特定位点。首先，通过新生链挤出法，然后通过蔗糖梯度沉降，从灵长类转化的和原代细胞系（HeLa细胞、原代人成纤维细胞、非洲绿色猴肾成纤维细胞[CV-1]和原代非洲绿色猴肾细胞）中分离新合成的含有复制起点的DNA。通过改进的最近邻分析，确定了新生和基因组DNA的所有四个可能的二核苷酸序列中的胞嘧啶甲基化水平。在新生和基因组DNA中观察到的胞嘧啶甲基化水平是相等的，表明DNA复制和甲基化是伴随事件。冈崎片段也被证明是甲基化的，这表明甲基化的快速动力学是新生DNA的前导链和滞后链的特征。然而，与先前的观察相反，新生DNA和基因组DNA都不包含在除了CpG之外的二核苷酸环境中可检测水平的甲基化胞嘧啶（即，CpA、CpC和CpT未甲基化）。最近邻分析还显示，相对于原代细胞系，癌细胞系在新生和基因组DNA中均高度甲基化。新生和基因组DNA在特定位点的甲基化程度也通过亚硫酸氢盐定位核纤层蛋白B2、c-myc和β-珠蛋白复制起点的CpG位点来确定。基因组和新生克隆的甲基化模式是相同的，证实了甲基化与复制同时发生的假设。有趣的是，在所有测试的克隆中发现c-myc起源未甲基化。这些结果表明，像基因一样，不同的复制起点表现出不同的甲基化模式。总之，我们的研究结果表明DNA甲基化和复制的紧密协调，这与最近的观察结果一致，表明DNA甲基转移酶与复制叉中的增殖细胞核抗原相关。
Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA, However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and beta-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-myc origin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.