Overreplication of short DNA regions during S phase in human cells
Overreplication of short DNA regions during S phase in human cells
复制标题
DOI：
10.1101/gad.445608
复制
发表时间：
2008-02-01
影响因子：
10.5
通讯作者：
Antequera, Francisco
中科院分区：
文献类型：
--
作者：
Gomez, Maria;Antequera, Francisco
DNA复制起点（ORI）是基因组在每个细胞周期复制一次的调控区域。在哺乳动物染色体中广泛使用的鉴定方法依赖于对候选区域的DNA新生链进行定量PCR。我们开发了一种新的高分辨率PCR策略，直接在未分离的人类DNA上定位ori。这种方法提供的灵敏度的提高揭示了一个类似200个碱基对重叠的具有良好特征的复制起始的短区域经历了几轮复制，与它们在S期的特定激活时间相一致。这一过程产生了一群离散的dsDNA片段，可以在正常增殖细胞的总DNA制备中作为自由分子检测到。过度复制区域在无核小体间隙边缘具有精确的边界，该间隙包含CpG岛启动子的转录起始位点。主动转录本身不会诱导过度复制，但会在与启动子相关的ORIs上刺激过度复制。短DNA片段的过量产生和ORI活性在时间和空间上的巧合预测了人类基因组中数千个ORI的精确定位，并揭示了DNA复制启动过程中一个以前未被注意到的步骤。
DNA replication origins (ORI) are regulatory regions from which the genome is replicated once every cell cycle. A widely used method for their identification in mammalian chromosomes relies on quantitative PCR of DNA nascent strands across candidate regions. We developed a new high-resolution PCR strategy to localize ORIs directly on total unfractionated human DNA. The increase in sensitivity provided by this approach has revealed that a short region of similar to 200-base-pair overlapping well-characterized replication origins undergoes several rounds of replication, coinciding with their specific time of activation during S phase. This process generates a population of discrete dsDNA fragments detectable as free molecules in preparations of total DNA in normally proliferating cells. Overreplicated regions have precise boundaries at the edge of the nucleosome-free gap that encompasses the transcription initiation sites of CpG island promoters. By itself, active transcription does not induce overreplication but does stimulate it at ORIs associated with promoters. The coincidence in time and space between the overproduction of short DNA fragments and ORI activity predicts the precise localization of thousands of ORIs in the human genome and uncovers a previously unnoticed step in the initiation of DNA replication.