Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus
Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus
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DOI：
10.1128/jb.181.7.1984-1993.1999
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发表时间：
1999-04-01
影响因子：
3.2
通讯作者：
Marczynski, GT
中科院分区：
文献类型：
--
作者：
Marczynski, GT
新月柄杆菌表现出对染色体复制和 DNA 甲基化的细胞类型特异性控制。不对称细胞分裂产生复制的茎细胞和非复制的群细胞。活动的群细胞必须分化成无柄细胞，以便复制和执行不对称细胞分裂。这种细胞分裂程序意味着染色体复制在每个细胞周期仅在有柄细胞中启动一次。 DNA 甲基化仅限于分裂前细胞阶段，并且由于 DNA 合成产生未甲基化的新生链，因此晚期 DNA 甲基化也意味着靠近复制起点的 DNA 比位于较远的 DNA 保持半甲基化的时间更长。在本报告中，这两个假设均通过基于 Tn5 的工程转座子 Tn5 Ohm-MP 进行了测试。这样可以进行灵敏的 Southern blot 检测，测量完全甲基化、半甲基化和未甲基化的 DNA 双链体。 Tn5 Ohm-MP 被放置在染色体周围的 11 个位点，并且清楚地证明靠近复制起点的 Tn5 Ohm-MP DNA 比位于更远的 DNA 保持半甲基化的时间更长。放置在复制起点附近的一个 Tn5 Ohm-MP 揭示了复制茎细胞中少量但可检测到的未甲基化双链 DNA。额外的 DNA 合成产生第二条未甲基化的新生链。因此，非甲基化 DNA 的测量是对 C. crescentus 染色体复制“每个细胞周期一次且仅一次”规则的关键测试。不到千分之一的有柄细胞过早启动第二轮染色体复制。我们讨论了细菌细胞周期对染色体复制的非常精确的负控制的影响。
Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5 Ohm-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5 Ohm-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5 Ohm-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5 Ohm-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.