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：
8600655
负责人：
Naoko Shima
金额：
$ 28.83万
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 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8600655
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

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 MCM2-7 复合物（复制的复制体）的结合获得许可。解旋酶，并且被调节为在 S 阶段仅激发一次。据信 MCM2-7 复合物存在于染色质的含量超过活跃复制起点的 10 倍以上，并且未使用的 MCM2-7 复合物是最常见的可能在 S 期被持续的复制叉从染色质中取代。这样做的原因染色质上 MCM2-7 复合物的过量仍然很大程度上未知。然而，当前的假设提出这些过量的 MCM2-7 复合物在正常 S 期中是可有可无的，但由于它们提供了备份起源，因此需要在扰动的 S 阶段中生存下来。在这里，我们建议这些备份源大量存在，因为它们的使用可以拯救以无重组的方式停滞叉子。此外，备份起源的这种抗重组作用是肿瘤抑制所需的，因为备份起源的约 50% 丢失会导致自发性肿瘤发生 Mcm4Chaos3/Chaos3 小鼠具有完全外显率。该提案的目标是阐明备份的作用使用 Mcm4Chaos3 小鼠进行肿瘤抑制的起源。以下是我们的具体目标。 1) 证明 Mcm4Chaos3/Chaos3 细胞中的备份源救援减少。使用备份源它们在叉恢复中的作用将通过称为 DNA 纤维的单分子分析进行测试。 2) 证明备份起源很少的染色体区域是脆弱的。通过结合染色质芯片免疫沉淀 (ChIP) 和细胞遗传学分析，我们将测试备份起始水平作为其中之一常见脆弱位点的决定因素，特定位点特别容易受到叉停顿的影响。 3）证明备份起源抑制同源重组和肿瘤发生。我们将确定备份起源在使用称为 FYDR 的转基因位点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- 中进行测试通过交换解决重组中间体的背景不足。 4) 测试备份起源和同源重组的丢失是否是合成致死的。测试关键备份源对分叉恢复的贡献，我们将确定备份源是否伴随受损拯救和同源重组导致合成致死和肿瘤抑制。预期结果将对我们对分叉路径选择的理解产生重大影响恢复和开发新的癌症疗法，因为备用起源的作用在很大程度上被忽视了。此外，预期的结果将证明复制起点的许可不足会导致癌症，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因果作用在癌症中观察到的不稳定性。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Helq acts in parallel to Fancc to suppress replication-associated genome instability.

DOI：
10.1093/nar/gkt676
发表时间：
2013-12
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Luebben SW;Kawabata T;Akre MK;Lee WL;Johnson CS;O'Sullivan MG;Shima N
通讯作者：
Shima N

Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression.

DOI：
10.1016/j.molcel.2011.02.006
发表时间：
2011-03-04
期刊：
Molecular cell
影响因子：
16
作者：
Kawabata T;Luebben SW;Yamaguchi S;Ilves I;Matise I;Buske T;Botchan MR;Shima N
通讯作者：
Shima N

A concomitant loss of dormant origins and FANCC exacerbates genome instability by impairing DNA replication fork progression.