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

摘要：起源许可确保真核细胞基因组在每个细胞周期内精确复制一次。 在G1期，复制起点通过与MCM2-7复合体的结合而获得许可，该复制物 解旋酶，并且在S阶段只被调节发射一次。据认为，MCM2-7络合物存在于 染色质比活性复制起点多10倍，未使用的MCM2-7复合体最多 在S阶段，可能被正在进行的复制分叉取代了染色质。这样做的原因是 染色质上MCM2-7复合体的过度丰度在很大程度上仍不清楚。然而，一股潮流 假说认为，这些过剩的MCM2-7络合物在正常的S相中是不必要的，但 需要在惊慌失措的S阶段生存下来，为他们提供后备来源。 在这里，我们认为这些备份来源大量存在，因为它们的使用允许救援 失速的叉子以一种无重组的方式。此外，备份起源的这种反重组作用是 肿瘤抑制所必需的，因为大约50%的备份来源的损失会导致自发的肿瘤形成 Mcm4Chaos3/Chaos3小鼠具有完全外显。这项提议的目标是解开备份的角色 起源于Mcm4Chaos3小鼠的肿瘤抑制。以下是我们的具体目标。 1)证明Mcm4Chaos3/Chaos3细胞的备份起源挽救减少。备份来源的使用 它们在叉子恢复中的作用将通过一种名为DNA纤维的单分子分析来测试。 2)证明备份来源少的染色体区域是脆弱的。通过结合染色质 免疫沉淀(芯片)和细胞遗传学分析，我们将检测备份来源水平作为其中之一 确定常见脆弱部位的决定因素，特别是对叉子失速特别敏感的基因座。 3)证明备份起源抑制同源重组和肿瘤发生。我们将决定 备份起源在使用名为FYDR的转基因基因座抑制非法重组中的作用。这个 过度同源重组在Mcm4Chaos3肿瘤发生中的作用将在BLM- 缺乏通过交换来分解重组中间体的背景。 4)检验备份来源的丢失和同源重组是否为合成致命性。要测试关键的 备份来源对分支恢复的贡献，我们将确定是否伴随备份来源的损害 挽救和同源重组导致合成杀伤力和肿瘤抑制。 预期的结果将对我们理解分叉的路径选择产生重大影响 恢复和开发新的癌症疗法，因为后备来源的作用在很大程度上被忽视了。 此外，预期的结果将表明，复制来源许可不足本身就会导致 癌症，强化了当前的观点，即放松管制的原产地许可对基因组具有致病作用 在癌症中观察到的不稳定性。

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.