5-azacytidine-induced Methyltransferase-DNA adducts block DNA replication in vivo
5-azacytidine-induced Methyltransferase-DNA adducts block DNA replication in vivo
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DOI：
10.1158/0008-5472.can-07-1038
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发表时间：
2007-09-01
期刊：
影响因子：
11.2
通讯作者：
Kreuzer, Kenneth N.
中科院分区：
文献类型：
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作者：
Kuo, H. Kenny;Griffith, Jack D.;Kreuzer, Kenneth N.
5-氮杂胞苷（aza-C）及其衍生物是用于白血病化疗的胞苷类似物。aza-C的主要作用是抑制胞嘧啶甲基化，这导致在胞嘧啶甲基化位点处形成共价甲基转移酶-DNA（MTase-DNA）加合物。这些加合物已被认为会导致染色体重排，并有助于细胞毒性，但详细的机制尚未阐明。我们使用二维琼脂糖凝胶电泳和电子显微镜分析质粒pBR 322在aza-C存在下生长的大肠杆菌细胞中的复制动力学。二维凝胶分析揭示了特定的气泡和Y分子的积累，依赖于胞嘧啶MTase EcoRII（M.EcoRII）的过度生产和aza-C的处理。此外，消除特定EcoRII甲基化位点的点突变导致相应的气泡和Y分子消失。这些结果意味着aza-C诱导的MTase-DNA加合物在体内阻断DNA复制。在aza-C处理后还观察到RecA依赖的X结构。这些分子可以通过重组修复和/或复制叉退化从阻断的叉产生。此外，电子显微镜分析揭示了aza-C处理后的气泡和滚动圈（RC）。这些结果表明，在复制叉被MTase-DNA加合物阻止后，复制可以从θ模式切换到RC模式。theta模式转化为RC模式的最简单模型是阻断的复制叉被分支特异性核酸内切酶切割。这种复制依赖性DNA断裂可能是导致基因组重排和/或细胞毒性的重要途径。
5-Azacytidine (aza-C) and its derivatives are cytidine analogues used for leukemia chemotherapy. The primary effect of aza-C is the prohibition of cytosine methylation, which results in covalent methyltransferase-DNA (MTase-DNA) adducts at cytosine methylation sites. These adducts have been suggested to cause chromosomal rearrangements and contribute to cytotoxicity, but the detailed mechanisms have not been elucidated. We used two-dimensional agarose gel electrophoresis and electron microscopy to analyze plasmid pBR322 replication dynamics in Escherichia coli cells grown in the presence of aza-C. Two-dimensional gel analysis revealed the accumulation of specific bubble and Y molecules, dependent on overproduction of the cytosine MTase EcoRII (M.EcoRII) and treatment with aza-C. Furthermore, a point mutation that eliminates a particular EcoRII methylation site resulted in disappearance of the corresponding bubble and Y molecules. These results imply that aza-C-induced MTase-DNA adducts block DNA replication in vivo. RecA-dependent X structures were also observed after aza-C treatment. These molecules may be generated from blocked forks by recombinational repair and/or replication fork regression. In addition, electron microscopy analysis revealed both bubbles and rolling circles (RC) after aza-C treatment. These results suggest that replication can switch from theta to RC mode after a replication fork is stalled by an MTase-DNA adduct. The simplest model for the conversion of theta to RC mode is that the blocked replication fork is cleaved by a branch-specific endonuclease. Such replication-dependent DNA breaks may represent an important pathway that contributes to genome rearrangement and/or cytotoxicity.