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

翻译后摘要：原产地许可，确保真核基因组复制精确每细胞周期一次。 在G1期，复制起点通过结合MCM 2 -7复合物，即复制起始点， 解旋酶，并且被调节为在S期期间仅发射一次。据信MCM 2 -7复合物存在于 染色质超过活性复制起点10倍，未使用的MCM 2 -7复合物是最常见的。 很可能在S期被正在进行的复制叉从染色质中置换出来。其原因 染色质上的MCM 2 -7复合物的过量仍然是未知的。然而，目前 一种假说提出，这些过量的MCM 2 -7复合物在正常的S期被抑制， 需要生存扰动的S阶段，因为他们提供备份的起源。 在这里，我们认为这些备份源大量存在，因为它们的使用允许拯救 以无重组的方式停止分叉。此外，备份来源的这种抗重组作用是 这是肿瘤抑制所必需的，因为约50%的备份来源丢失会导致自发性肿瘤发生， Mcm 4Chaos 3/Chaos 3小鼠，具有完全免疫抑制。本提案的目标是阐明备份的作用 使用Mcm 4Chaos 3小鼠的肿瘤抑制起源。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 它们在叉子恢复中的作用将用一种称为DNA纤维的单分子分析来测试。 2)证明几乎没有后备来源的染色体区域是脆弱的。通过结合染色质 芯片上的免疫沉淀（ChIP）和细胞遗传学分析，我们将测试备份的起源水平作为其中一个 决定因素为共同的脆弱网站，具体的位点特别容易叉失速。 3)证明备用起源抑制同源重组和肿瘤发生。我们将确定 利用一个叫做FYDR的转基因位点，研究备用基因在抑制非法重组中的作用。的 过量同源重组对Mcm 4Chaos 3肿瘤发生的贡献将在BLM中测试， 缺乏背景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。为了测试关键的 备份源对fork恢复的贡献，我们将确定备份源的伴随损害 拯救和同源重组导致合成致死和肿瘤抑制。 预期的结果将对我们理解分叉的途径选择产生重大影响 然而，癌症的治疗和康复以及开发新的癌症疗法，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，仅复制起点的许可不足就会导致 癌症，加强了目前的观点，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.