Replication fork density increases during DNA synthesis in X-laevis egg extracts
Replication fork density increases during DNA synthesis in X-laevis egg extracts
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DOI：
10.1006/jmbi.2000.3930
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发表时间：
2000-07-28
影响因子：
5.6
通讯作者：
Bensimon, A
中科院分区：
文献类型：
--
作者：
Herrick, J;Stanislawski, P;Bensimon, A
真核生物基因组的复制依赖于细胞周期中DNA复制的时间和空间组织。为了研究高等真核生物中DNA复制的基因组组织，随着DNA合成在细胞周期的S期进行，必须同时分析基因组大区域的多个复制起点。我们采用了一种新技术，可以在全基因组的基础上定量分析DNA复制。这项技术涉及在玻璃表面拉伸和排列单个DNA分子。作为一个模型系统，非洲爪蟾卵提取物被用来差异标记精子染色质在连续的时间点后开始的DNA合成。差异标记的DNA允许区分更早和更晚的复制序列，因此可以直接可视化任何给定时间的DNA合成位点。提取基因组DNA，对线性化分子进行测量，提供了X染色体中DNA复制的空间和时间组织的综合分析。体外复制系统。结果发现：（1）DNA合成以不规则的时间间隔异步启动，但随着DNA复制的进展，这种启动是连续的;（2）DNA合成接近完成时，启动的频率（每种酶激活的起始点的数量）增加。这些研究结果的影响，在早期胚胎的DNA复制的调节进行了讨论。(C)北京大学出版社.
Duplication of the eukaryotic genome depends on the temporal and spatial organization of DNA replication during the cell cycle. To investigate the genomic organization of DNA replication in a higher eukaryote, multiple origins of replication must be simultaneously analyzed over large regions of the genome as DNA synthesis progresses through S phase of the cell cycle. We have employed a novel technique that allows for the quantitative analysis of DNA replication on a genome wide basis. The technique involves stretching and aligning individual DNA molecules on a glass surface. As a model system, Xenopus laevis egg extract was used to differentially label sperm chromatin at successive time points after the start of DNA synthesis. The differentially labeled DNA allows earlier and later replicating sequences to be distinguished, and hence the sites of DNA synthesis at any given time can be directly visualized. Genomic DNA was extracted, and measurements made on the linearized molecules provided a comprehensive analysis of the spatial and temporal organization of DNA replication in the X. laevis in vitro replication system. It was found that: (i) DNA synthesis initiates asynchronously at irregular intervals but continuously as DNA replication advances; and (ii) that the frequency of initiation (the number of activated origins per kilobase) increases as DNA synthesis nears completion. The implications of these findings for the regulation of DNA replication in early embryos is discussed. (C) 2000 Academic Press.