Department of Medicine and Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Genome Res. 2009 Dec;19(12):2288-99. doi: 10.1101/gr.094060.109. Epub 2009 Sep 18.
The organization of mammalian DNA replication is poorly understood. We have produced high-resolution dynamic maps of the timing of replication in human erythroid, mesenchymal, and embryonic stem (ES) cells using TimEX, a method that relies on gaussian convolution of massive, highly redundant determinations of DNA copy-number variations during S phase to produce replication timing profiles. We first obtained timing maps of 3% of the genome using high-density oligonucleotide tiling arrays and then extended the TimEX method genome-wide using massively parallel sequencing. We show that in untransformed human cells, timing of replication is highly regulated and highly synchronous, and that many genomic segments are replicated in temporal transition regions devoid of initiation, where replication forks progress unidirectionally from origins that can be hundreds of kilobases away. Absence of initiation in one transition region is shown at the molecular level by single molecule analysis of replicated DNA (SMARD). Comparison of ES and erythroid cells replication patterns revealed that these cells replicate about 20% of their genome in different quarters of S phase. Importantly, we detected a strong inverse relationship between timing of replication and distance to the closest expressed gene. This relationship can be used to predict tissue-specific timing of replication profiles from expression data and genomic annotations. We also provide evidence that early origins of replication are preferentially located near highly expressed genes, that mid-firing origins are located near moderately expressed genes, and that late-firing origins are located far from genes.
哺乳动物 DNA 复制的组织方式还不太清楚。我们利用 TimEX 方法生成了人类红系、间充质和胚胎干细胞（ES 细胞）中复制时间的高分辨率动态图谱，该方法依赖于在 S 期大量高度冗余的 DNA 拷贝数变化测定值的高斯卷积，以产生复制时间图谱。我们首先使用高密度寡核苷酸平铺阵列获得了基因组 3%的时间图谱，然后使用大规模并行测序方法将 TimEX 方法扩展到全基因组范围。我们表明，在未转化的人类细胞中，复制时间受到高度调控且高度同步，许多基因组片段在没有起始的时间转换区中复制，其中复制叉从数百千碱基远的起始点单向推进。通过复制 DNA 的单分子分析（SMARD）在分子水平上显示了一个转换区中没有起始。ES 和红系细胞复制模式的比较表明，这些细胞在 S 期的不同四分之一中复制了大约 20%的基因组。重要的是，我们检测到复制时间与离最近表达基因的距离之间存在很强的负相关关系。这种关系可用于根据表达数据和基因组注释来预测组织特异性的复制时间图谱。我们还提供了证据表明，早期复制起始点优先位于高度表达基因附近，中频起始点位于中度表达基因附近，而低频起始点则远离基因。
