Backup origins: their role in replication fork recovery and tumor suppression

描述（由申请人提供）：摘要：原产地许可确保真核基因组在每个细胞周期精确复制一次。在G1期，复制起点通过结合MCM 2 -7复合物（复制解旋酶）获得许可，并在S期被调节为仅发射一次。据信，MCM 2 -7复合物以超过活性复制起点的>10倍过量存在于染色质上，并且未使用的MCM 2 -7复合物最有可能在S期期间被正在进行的复制叉从染色质置换。染色质上的MCM 2 -7复合物过多的原因在很大程度上仍然未知。然而，目前的假设提出，这些过量的MCM 2 -7复合物在正常的S期被抑制，但需要在扰动的S期存活，因为它们提供了备用来源。 在这里，我们建议，这些备份的起源存在丰富的，因为它们的使用允许救援停滞的叉在重组自由的方式。此外，肿瘤抑制需要备用来源的这种抗重组发生作用，因为备用来源的~50%损失导致具有完全转移的Mcm 4Chaos 3/Chaos 3小鼠中的自发肿瘤发生。该提案的目标是使用Mcm 4Chaos 3小鼠阐明备用起源在肿瘤抑制中的作用。以下是我们的具体目标。 1)证明Mcm 4Chaos 3/Chaos 3单元中的备份起源救援减少。备份源的使用及其在分叉恢复中的作用将使用称为DNA纤维的单分子分析进行测试。 2)证明几乎没有后备来源的染色体区域是脆弱的。通过结合芯片上的染色质免疫沉淀（ChIP）和细胞遗传学分析，我们将测试备份的起源水平作为常见的脆弱位点，特别是易受叉失速的特定位点的决定因素之一。 3)证明备用起源抑制同源重组和肿瘤发生。我们将使用一个名为FYDR的转基因位点来确定后备来源在抑制非法重组中的作用。将在BLM缺陷背景中测试过度同源重组对Mcm 4Chaos 3肿瘤发生的贡献，以通过交换解析重组中间体。 4)测试备份起源和同源重组的丢失是否是合成致死。为了测试备份起源对分叉恢复的关键贡献，我们将确定备份起源拯救和同源重组的伴随损害是否导致合成致死率和肿瘤抑制。 预期的结果将对我们理解分叉恢复的途径选择和开发新的癌症疗法产生重大影响，因为备份起源的作用在很大程度上被忽视了。此外，预期的结果将表明，单独的复制起点许可不足会导致癌症，加强目前的观点，即放松管制的起点许可对癌症中观察到的基因组不稳定性具有致病作用。 公共卫生相关性：项目叙述：该应用程序侧重于癌症发展早期可能发生的事件。该提案的目的是为开发新的癌症疗法提供信息。

DESCRIPTION (provided by applicant): Abstract: Origin licensing ensures that the eukaryotic genome is replicated precisely once per cell cycle. During the G1 phase, replication origins are licensed by the binding of MCM2-7 complexes, the replicative helicases, and are regulated to fire only once during S phase. It is believed that MCM2-7 complexes exist on chromatin in a >10-fold excess over active replication origins, and that unused MCM2-7 complexes are most likely displaced from the chromatin by ongoing replication forks during S phase. The reason for this overabundance of the MCM2-7 complexes on chromatin has remained largely unknown. However, a current hypothesis proposes that these excess MCM2-7 complexes are dispensable in normal S phase but are required to survive perturbed S phase as they provide backup origins. Here, we propose that these backup origins exist abundantly because their use allows for the rescue of stalled forks in a recombination-free manner. Moreover, this anti-recombinogenic role of backup origins is required for tumor suppression, because a ~50% loss of backup origins leads to spontaneous tumorigenesis in Mcm4Chaos3/Chaos3 mice with complete penetrance. The goal of this proposal is to unravel the role of backup origins in tumor suppression using Mcm4Chaos3 mice. The following are our specific aims. 1) Demonstrate that backup origin rescue is reduced in Mcm4Chaos3/Chaos3 cells. The use of backup origins and their role in fork recovery will be tested using a single molecule analysis called DNA fiber. 2) Demonstrate that chromosome regions with few backup origins are fragile. By combining chromatin immunoprecipitation (ChIP) on chip and cytogenetic analyses, we will test backup origin levels as one of the determining factors for common fragile sites, specific loci particularly susceptible to fork stalling. 3) Prove that backup origins suppress homologous recombination and tumorigenesis. We will determine the role of backup origins in suppressing illegitimate recombination using a transgenic locus called FYDR. The contribution of excessive homologous recombination to Mcm4Chaos3 tumorigenesis will be tested in a BLM- deficient background to resolve recombination intermediates via crossing-over. 4) Test if the loss of backup origins and homologous recombination is synthetic lethal. To test the critical contribution of backup origins to fork recovery, we will determine if concomitant impairment of backup origin rescue and homologous recombination results in synthetic lethality and tumor suppression. The expected outcomes would have a significant impact on our understanding of pathway choice for fork recovery and developing new cancer therapies, as the role of backup origins has been largely ignored. Furthermore, the expected results will demonstrate that under-licensing of replication origins alone causes cancer, strengthening the current view that deregulated origin licensing has a causative role for the genome instability observed in cancer. PUBLIC HEALTH RELEVANCE: Project Narrative: This application focuses on the events that may happen in very early stages of cancer development. The goal of this proposal is to provide information for the development of new cancer therapies.