Mapping replication elements on human chromosomes

描述：(申请人提供)在细胞周期的脱氧核糖核酸合成(S)阶段，脱氧核糖核酸复制的起始点在人类染色体上的离散位置。在这些起始点开始的复制分叉双向移动，并在遇到来自相反方向的分叉时终止。起始区和终止区的身份在人类染色体中知之甚少。此外，在S相开始时，所有的起源并不是同步激发的。火源有的早于S期，有的晚于S期。关于人类染色体上这种早激发和晚激发起源的分布，以及它们相对于活跃转录基因的位置，目前还知之甚少。在本项目中，使用实验室验证的方法，将从从G1-S边界同步释放的HeLa细胞中提纯在特定时间在S期复制的DNA。这些定时复制产品将被用作DNA片段微阵列的探针，这些DNA片段代表了本次RFA的目标--人类基因组的30MB。DNA标记的复制时间与物理位置的关系图将允许识别起始点，包括早期和晚期，因为起始点特定的片段将比相邻片段复制得更早。该图还将揭示向相反方向移动的复制叉子在哪里相遇(或终止)。假设的起始点两侧的直线斜率将是复制叉子移动速度的量度。通过对S期细胞复制中间产物的中性/中性双向凝胶电泳法，将含有片段的起始子集确认为真正的起始物。这一分析将允许区分单个地点的起源和包含一群起源的起始区。总而言之，结果将确定重要的复制要素：起点、起始区和终止点。此外，他们还将识别染色体上具有复制暂停位置的位置和/或复制配置文件突然改变的区域。

DESCRIPTION: (provided by applicant) During the DNA synthesis (S) phase of the cell-cycle, origins of DNA replication fire at discrete sites on human chromosomes. The replication forks that initiate at these origins move bi-directionally and terminate when they encounter forks coming from the opposite direction. The identities of the origins and termination zones are poorly understood in human chromosomes. In addition, all origins do not fire synchronously at the onset of S phase. Some origins fire early in S phase while others fire late in S phase. Not much is know about the distribution of such early and late firing origins on human chromosomes and their location relative to actively transcribed genes. In this project, using methods validated in the laboratory, DNA replicated at specific times in S phase will be purified from HeLa cells synchronously released from the G1-S boundary. These timed-replication products will be used as probes on microarrays of DNA fragments representing the 30 mB of the human genome that is the target of this RFA. A plot of the time of replication versus physical location of the DNA markers will allow the identification of origins, both early and late, because origin specific segments will replicate earlier than adjoining segments. The plot will also reveal where replication forks moving in opposite directions meet (or terminate). The slope of the line on either side of putative origins will be a measure of the speed of replication fork movement. A subset of the origin containing fragments will be confirmed to be bona-fide origins by neutral/neutral 2D gel electrophoresis of replication intermediates harvested from cells at the time in S phase when these origins are expected to initiate. This analysis will allow the distinction of single site origins versus initiation zones containing a cluster of origins. Collectively the results will identify important replication elements: origins, initiation zones and termination sites. In addition, they will identify sites in the chromosomes with replication pause sites and/or zones where replication profiles change abruptly.