Effect of anti-S phase agents on human chromosomes

描述（由申请人提供）：抗S期药物如阿霉素、吉西他滨、阿糖胞苷和羟基脲广泛用于治疗癌症和血液学疾病，如原发性血小板减少症、真性红细胞增多症和镰状细胞性贫血。所有这些试剂被认为引起复制叉停滞，导致抑制细胞增殖的检查点途径的激活。然而，关于这些抑制剂如何抑制复制起点、负责检查点激活的位点/DNA损伤以及这些损伤对染色体结构或基因表达的影响，知之甚少。此外，已知许多这些药剂通过未知机制影响基因表达。例如，羟基脲已被用于镰状细胞性贫血，因为它增加了胎儿血红蛋白的表达，降低了细胞表面粘附分子的表达。 我们最近发表了研究复制时间、复制起点和抗S期试剂对人类细胞中复制起点的影响的基因组方法。我们已经发现，用于治疗的两种抗S期试剂导致成簇的新起点的激发，导致基因组中限定位点处的高密度停滞复制叉。 在这项提案中，我们计划确定这是否是其他抗S期药物的一般特征，这些聚集的新起源发生的位点是否在不同的细胞系中相同，或者当细胞在S期的所有部分暴露于这些药物时，这些位点是否发生变化。我们将研究是否在聚集的新起源的高密度的停滞复制叉使这些网站的基因组易受双链断裂（DSB）和染色体重排。我们将研究由于成簇的新起源的高密度引起的染色质的干扰状态是否导致基因表达的抑制。最后，我们将研究哪些，在哪里和以什么顺序检查点激活剂和DNA修复蛋白被招募到停滞的复制叉创建的抗S期代理。

DESCRIPTION (provided by applicant): Anti-S phase agents like doxorubicin, gemcitabine, cytarabine and hydroxyurea are in extensive use for the treatment of cancers and of hematological diseases such as essential thrombocytopenia, polycythemia vera and sickle cell anemia. All these agents are believed to cause replication fork stalling leading to activation of checkpoint pathways that inhibit cell proliferation. Yet, not much is known about how these inhibitors inhibit the origins of replication, of the sites/DNA lesions responsible for checkpoint activation and of the effect of these lesions on chromosome structure or gene expression. In addition, many of these agents are known to affect gene expression by unknown mechanisms. For example, hydroxyurea has been used in sickle cell anemia because it increases the expression of fetal hemoglobin and decreases the expression of cell-surface adhesion molecules. We have recently published genomic approaches to study time of replication, origins of replication and effects of anti-S phase agents on the replication origins in human cells We have discovered that two anti-S phase agents used for therapy lead to the firing of clustered neo-origins, leading to a high density of stalled replication forks at defined sites in the genome. In this proposal we plan to determine whether this is a general feature of other anti-S phase agents, whether the sites where these clustered neo-origins occur are the same in different cell lines or whether the sites change when cells are exposed to these agents in all parts of the S phase. We will investigate whether the high density of stalled replication forks at clustered neo-origins make these sites of the genome susceptible to double-strand breaks (DSB) and chromosomal rearrangements. We will examine whether the disturbed state of the chromatin due to the high density of clustered neo-origins leads to suppression of gene expression. Finally, we will examine which, where and in what order checkpoint activators and DNA repair proteins are recruited to the stalled replication forks created by anti-S phase agents.