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

备份起源：它们在复制叉恢复和肿瘤抑制中的作用

基本信息

批准号：
8403816
负责人：
Naoko Shima
金额：
$ 27.96万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8403816
关键词：
Automobile Driving Binding Cancer Etiology Cell Cycle Cells Chromatin Chromosome Fragile Sites Chromosomes Complex Copy Number Polymorphism Cytogenetic Analysis DNA DNA Sequence Rearrangement DNA biosynthesis Development Diagnostic Neoplasm Staging Ensure Event Fiber Fire - disasters G1 Phase Genes Genetic Crossing Over Genetic Recombination Genome Genomic Instability Goals Impairment Knowledge Licensing Loss of Heterozygosity MCM2 gene Malignant Neoplasms Mediating Mus Mutant Strains Mice Outcome Pathway interactions Penetrance Play Process Recovery Replication Origin Risk Role S Phase Testing Transgenic Organisms Travel Tumor Suppression abstracting cancer prevention cancer therapy chromatin immunoprecipitation helicase homologous recombination human disease innovation mammalian genome prevent single molecule tumor tumorigenesis

Automobile Driving Binding Cancer Etiology Cell Cycle Cells Chromatin Chromosome Fragile Sites Chromosomes Complex Copy Number Polymorphism Cytogenetic Analysis DNA DNA Sequence Rearrangement DNA biosynthesis Development Diagnostic Neoplasm Staging Ensure Event Fiber Fire - disasters G1 Phase Genes Genetic Crossing Over Genetic Recombination Genome Genomic Instability Goals Impairment Knowledge Licensing Loss of Heterozygosity MCM2 gene Malignant Neoplasms Mediating Mus Mutant Strains Mice Outcome Pathway interactions Penetrance Play Process Recovery Replication Origin Risk Role S Phase Testing Transgenic Organisms Travel Tumor Suppression abstracting cancer prevention cancer therapy chromatin immunoprecipitation helicase homologous recombination human disease innovation mammalian genome prevent single molecule tumor tumorigenesis

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项目摘要

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.

摘要：起源许可确保每个细胞周期精确地复制真核基因组。在G1阶段，复制起源是由MCM2-7复合物的结合（复制）许可的解旋酶，并在S阶段只能发射一次。据信MCM2-7复合物存在与主动复制起源相比，染色质大于10倍，而未使用的MCM2-7复合物是最多的在S相期间，可能通过持续的复制叉从染色质移位。原因 MCM2-7复合物在染色质上的过度不足以在很大程度上未知。但是，电流假设提出这些过量的MCM2-7复合物在正常阶段是可分配的，但在提供备份起源时，必须在扰动的阶段生存。在这里，我们建议这些备份起源存在丰富，因为它们的使用允许营救以无重组方式停滞叉子。此外，备份起源的这种抗归因于成分作用是抑制肿瘤所需 MCM4CHAOS3/CHAOS3小鼠具有完整的渗透率。该提议的目的是揭开备份的作用使用MCM4CHAOS3小鼠抑制肿瘤的起源。以下是我们的具体目标。 1）证明MCM4CHAOS3/CHAOS3细胞中备份起源的救援降低。备用起源的使用并且将使用称为DNA纤维的单分子分析来测试它们在叉恢复中的作用。 2）证明备份起源很少的染色体区域是脆弱的。通过组合染色质在芯片和细胞遗传学分析中，免疫沉淀（芯片），我们将测试备份起源水平作为其中之一确定普通脆弱位点的因素，特定的基因座特别容易受到叉档的影响。 3）证明备用起源抑制了同源重组和肿瘤发生。我们将确定备用起源在使用称为Fydr的转基因基因座抑制非法重组中的作用。这过度同源重组对MCM4CHAOS3肿瘤发生的贡献将在BLM中进行测试通过交叉越过解决重组中间体的背景不足。 4）测试备用起源的丧失和同源重组是否是合成的致命。测试关键备用起源对叉恢复的贡献，我们将确定备份起源是否同时损害救援和同源重组会导致合成的致死性和抑制肿瘤。预期的结果将对我们对叉子选择的理解产生重大影响恢复和发展新的癌症疗法，因为备份起源的作用在很大程度上被忽略了。此外，预期的结果将表明，仅复制起源的许可不足会导致癌症，加强了当前的观点，即放松管制的起源许可对基因组具有致病作用在癌症中观察到不稳定性。