Mapping replication elements on human chromosomes

描述：（由申请人提供）在细胞周期的DNA合成(S)阶段，DNA复制的起源在人类染色体上的离散位点进行。在这些起点启动的复制分叉是双向移动的，当它们遇到来自相反方向的分叉时终止。在人类染色体中，起源区和终止区身份的了解很少。此外，在S期开始时，并非所有的起源都是同步的。一些起源在S期早期激发，而另一些在S期后期激发。对于人类染色体上这些早期和晚期激活起源的分布以及它们相对于活跃转录基因的位置，我们所知不多。在本项目中，使用实验室验证的方法，将从G1-S边界同步释放的HeLa细胞中纯化S期特定时间复制的DNA。这些定时复制产物将被用作DNA片段微阵列上的探针，这些DNA片段代表30mb的人类基因组，这是RFA的目标。复制时间与DNA标记物的物理位置的关系图将使起源的早期和晚期识别成为可能，因为起源特定的片段将比相邻的片段更早地复制。该图还将显示向相反方向移动的复制分叉在哪里相遇（或终止）。假定起点两侧的直线斜率将是复制叉移动速度的度量。含有片段的起源的一个子集将被确认为真正的起源，通过中性/中性2D凝胶电泳从S期细胞中收集复制中间体，当这些起源预计开始时。这种分析可以区分单位点起始区和包含一簇起始区。总的来说，结果将确定重要的复制元素：起源，起始区和终止位点。此外，它们将识别染色体中具有复制暂停位点和/或复制谱突变区域的位点。

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.