Interaction between DNMT1 and DNA replication reactions in the SV40 in vitro replication system
Interaction between DNMT1 and DNA replication reactions in the SV40 in vitro replication system
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DOI：
10.1111/j.1349-7006.2008.00913.x
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发表时间：
2008-10
期刊：
影响因子：
5.7
通讯作者：
Shintaro Shimamura;F. Ishikawa
中科院分区：
文献类型：
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作者：
Shintaro Shimamura;F. Ishikawa
与正常细胞相比，癌细胞表现出遗传和表观遗传的不稳定性。这些独特的性质在给定的癌细胞群体中产生了遗传和表观遗传的异质性，并为该群体通过克隆选择进行表型进化提供了一种手段。CpG二核苷酸的DNA甲基化是癌细胞中经常受到干扰的表观遗传标志之一。为了了解CpG甲基化模式在癌细胞中是如何变化的，有必要知道它是如何在正常细胞增殖中忠实地保持的。为了实现这一目标，我们开发了一种新的体外系统，该系统基于成熟的SV40体外复制系统，并具有重建并行DNA复制和DNA维持甲基化反应的功能。我们发现只有当外源DNA甲基转移酶1和S-腺苷蛋氨酸加入到反应中时，DNA甲基化才能保持。我们证明了DNMT1与复制和/或复制的染色质有关，与模板DNA的DNA甲基化状态无关。此外，DNMT1的增殖细胞核抗原结合域(PBD)不是关联所必需的。综上所述，我们认为DNMT1是以一种独立于DNA甲基化反应的结构性方式被招募来复制和/或复制染色质。这份报告中描述的体外系统对于分析DNA维持甲基化反应的分子机制非常有用。(《癌症科学》2008；99：1960-1966)
In contrast to normal cells, cancer cells exhibit both genetic and epigenetic instability. These unique properties give rise to genetic and epigenetic heterogeneity in a given population of cancer cells and provide a means for the population to undergo phenotypic progression by clonal selection. DNA methylation at CpG dinucleotides is one of the epigenetic marks that are frequently disturbed in cancer cells. To understand how the CpG methylation pattern is changeable in cancer cells, it is necessary to know how it is faithfully maintained in normal cell proliferation. Toward this goal, we have developed a novel in vitro system that is based on the well‐established SV40 in vitro replication system and functions to reconstitute concurrent DNA replication and DNA maintenance methylation reactions. We found that DNA methylation was maintained only when exogenous DNA methyltransferase 1 (DNMT1) and S‐adenosyl methionine (SAM) were added to the reaction. We demonstrated that DNMT1 associates with replicating and/or replicated chromatin irrespective of the DNA methylation status of template DNA. Moreover, the PCNA‐binding domain (PBD) of DNMT1 is not required for the association. Taken together, we suggest that DNMT1 is recruited to replicating and/or replicated chromatin in a constitutive manner independent of the DNA methylation reaction. The in vitro system described in this report is very useful for analyzing the molecular mechanism underlying the DNA maintenance methylation reaction. (Cancer Sci 2008; 99: 1960–1966)