Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS One. 2011;6(5):e17308. doi: 10.1371/journal.pone.0017308. Epub 2011 May 13.
Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.
METHODOLOGY/PRINCIPAL FINDINGS: We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7-1.5 kb obtained from the breast cancer cell line MCF-7 hybridized to a custom tiling array containing 50-60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5'ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.
CONCLUSIONS/SIGNIFICANCE: Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors.
哺乳动物基因组的复制需要激活数千个复制原点，这些复制原点在空间和时间上受到未知机制的调节。在最基本的层面上，我们对每个染色体、不同细胞类型中的复制原点的分布模式以及细胞的生理状态是否改变这种分布模式的了解非常有限。
方法/主要发现：我们使用了来自乳腺癌细胞系 MCF-7 的标准 λ-外切核酸酶抗性新生 DNA 制备物，该制备物的大小范围为 0.7-1.5 kb，与包含 50-60 nt 探针的定制平铺阵列杂交，这些探针均匀分布在覆盖人类基因组约 1%的基因和非基因区域中。类似的 DNA 制备物用于高通量 DNA 测序。还使用来自 BT-474 和 H520 细胞系的 DNA 进行了阵列实验。通过确定显示新生 DNA 富集的位点，我们定位了数千个 DNA 复制原点。我们的主要发现是：(a) 阵列和 DNA 测序实验方法产生的起源分布图谱基本相同；(b) 起源分布在所有测试的细胞系中基本保持不变（>70%）；(c) 起源在表达基因的 5'端和进化上保守的基因间序列中富集；(d) MCF-7 中的 ChIP-on-chip 实验表明，H3K4Me3 和 RNA 聚合酶 II 染色质结合位点在 DNA 复制原点处富集。
结论/意义：我们的结果表明，起源激活程序在不同的细胞类型中基本保持不变。此外，我们的工作支持了最近将转录起始与复制联系起来的研究，并进一步表明进化上保守的基因间序列有可能参与起源选择。总体而言，我们的观察结果表明，复制原点选择是一个依赖于局部复制因子可及性的随机过程。
