Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius
Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius
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DOI：
10.1073/pnas.0806414105
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发表时间：
2008-10-28
影响因子：
11.1
通讯作者：
Bell, Stephen D.
中科院分区：
文献类型：
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作者：
Duggin, Iain G.;McCallum, Simon A.;Bell, Stephen D.
“婴儿机器”提供了一种产生最小扰动细胞的同步培养物的方法。我们描述了使用该技术来建立硫化叶菌属超嗜热古菌的关键细胞周期参数。通过 2D 凝胶分析，将酸热硫化叶菌的 3 个 DNA 复制起点定位到 2,226 kb 环状基因组上的 0 (oriC2)、579 (oriC1) 和 1,197 kb (oriC3) 附近，并且我们直接证明了它们在同步细胞周期的最初几分钟内的活性。我们还在对数期细胞的起点处检测到了 X 形 DNA 分子，但这些分子与同步细胞中的复制起始或正在进行的染色体复制没有直接关系。同步和对数期培养物的全基因组标记频率分析表明，每轮复制的所有 3 个起点的起点利用率接近 100%。然而，与 oriC1 和 oriC3 相比，oriC2 的激活平均稍晚一些。在同步培养中，DNA 复制叉以大约每秒 88 bp 的速度双向远离每个起点。对 3 个 Orc1/Cdc6 起始蛋白的分析表明，整个细胞周期中细胞丰度和起源结合具有一致性。相反，尽管 MCM 解旋酶的水平在整个细胞周期中保持恒定，但其起源定位受到调节，因为它在早期 S 期的所有 3 个起源处都强烈富集。
The "baby machine" provides a means of generating synchronized cultures of minimally perturbed cells. We describe the use of this technique to establish the key cell-cycle parameters of hyperthermophilic archaea of the genus Sulfolobus. The 3 DNA replication origins of Sulfolobus acidocaldarius were mapped by 2D gel analysis to near 0 (oriC2), 579 (oriC1), and 1,197 kb (oriC3) on the 2,226-kb circular genome, and we present a direct demonstration of their activity within the first few minutes of a synchronous cell cycle. We also detected X-shaped DNA molecules at the origins in log-phase cells, but these were not directly associated with replication initiation or ongoing chromosome replication in synchronized cells. Whole-genome marker frequency analyses of both synchronous and log-phase cultures showed that origin utilization was close to 100% for all 3 origins per round of replication. However, oriC2 was activated slightly later on average compared with oriC1 and oriC3. The DNA replication forks moved bidirectionally away from each origin at approximate to 88 bp per second in synchronous culture. Analysis of the 3 Orc1/Cdc6 initiator proteins showed a uniformity of cellular abundance and origin binding throughout the cell cycle. In contrast, although levels of the MCM helicase were constant across the cell cycle, its origin localization was regulated, because it was strongly enriched at all 3 origins in early S phase.