The genome-wide DNA replication program in fission yeast

描述（由申请人提供）：尽管在过去的二十年中在理解真核细胞中DNA复制起始的机制方面取得了很大进展，但我们仍然不知道是什么控制了S期不同起始点的启动时间。这是一个重要的问题，因为不适当地调节复制时间与基因组不稳定性和癌症有关。 在此，纽约州布法罗罗斯威尔公园癌症研究所的Joel Huberman博士和斯托尼布鲁克纽约的州立大学的Janet Leatherwood博士的实验室提出利用最近完成的模式真核生物体裂殖酵母裂殖酵母的基因组序列，在野生型和突变型裂殖酵母细胞中进行基于微阵列的全基因组复制定时分析。我们将利用Leatherwood博士开发的裂变酵母全基因组微阵列分析的新设备，我们还将利用两个实验室在理解复制时序控制方面取得的早期进展。 拟议研究的结果将使我们能够确定复制起点的位置，它们的发射效率以及它们发射的时间。通过比较野生型细胞中早期和晚期点火起点的DNA序列，我们可能能够识别对复制时序控制重要的序列基序。通过比较每个基因组区域的复制时间与其基因表达水平，我们将发现在分裂酵母中复制时间与基因表达之间是否存在相关性。通过比较野生型细胞和携带特定突变基因的细胞之间的起始激发时间，我们将评估每个测试基因对于控制复制时间的重要性。我们打算把重点放在控制组蛋白乙酰化的基因和控制DNA损伤和复制应激的S期检查点反应的基因的作用。

DESCRIPTION (provided by applicant): Although great progress has been made in the past two decades in understanding the mechanism of initiation of DNA replication in eukaryotic cells, we still do not know what controls the times at which different origins fire during S phase. This is an important issue, because improperly regulated replication timing is associated with genomic instability and with cancer. Here the laboratories of Dr. Joel Huberman at Roswell Park Cancer Institute, Buffalo, NY, and of Dr. Janet Leatherwood at the State University of New York, Stony Brook, NY, propose to take advantage of the recently completed genome sequence of the model eukaryotic organism, the fission yeast Schizosaccharomyces pombe, to enable microarray-based genome-wide replication timing analyses in wild-type and mutant fission yeast cells. We shall utilize the new facility for fission yeast genome-wide microarray analyses developed by Dr. Leatherwood, and we shall also take advantage of earlier progress made by both laboratories toward understanding replication timing control. The results of the proposed studies will allow us to determine the locations of replication origins, their firing efficiencies, and the times at which they fire. By comparing the DNA sequences of early- and late-firing origins in wild-type cells, we may be able to identify sequence motifs important for replication timing control. By comparing the replication timing of each genomic region with its level of gene expression, we shall find out whether or not there are correlations between replication timing and gene expression in fission yeast. By comparing origin firing times between wild type cells and cells bearing specific mutated genes, we shall evaluate the importance of each tested gene for control of replication timing. We intend to focus on the roles of genes that control histone acetylation and genes that control S-phase checkpoint responses to DNA damage and replication stress.