Replication Licensing in Genome Stability, Cancer and Aging

描述(由申请人提供)：细胞分裂过程中DNA的准确复制是维持基因组完整性的关键，不准确的复制会导致基因组损伤导致癌症和其他与年龄相关的功能障碍。这些研究将提供加强准确DNA复制所需机制的方法，并预测当这些机制失效时可能发生损害的位置。该项目基于该实验室开发的转基因小鼠品系，该品系携带一种微妙的突变，导致复制机制的核心组件McM2表达不足，事实证明，这对于理解复制起源许可不足的后果非常有价值。这些小鼠非常容易患癌症，并在129Sv的遗传背景上显示出胸腺淋巴母细胞淋巴瘤的完全外显性。该项目支持的研究表明，在复制分叉停滞和崩溃的条件下，从原始起源和在更大程度上处于休眠(备份)起源的复制的低效启动是造成这种易感性的主要原因。此外，由此产生的肿瘤中的遗传损伤是以短缺失的形式出现的，这显著地有助于识别肿瘤抑制基因。此外，这些研究表明，某些品系的小鼠对McM2缺乏的后果的处理能力不同，这一点从肿瘤发生的减少中得到了证明。拟议的研究解决了三个关键问题：1)DNA复制启动位置的序列特异性在多大程度上直接或作为表观遗传修饰的结果，影响McM2缺陷小鼠复制相关遗传损伤可能发生的位置？2)缓解McM2缺陷引起的疾病表型的遗传因素是否通过影响特定位置的起源许可效率发挥作用，或者是否有替代机制来抑制这些菌株复制相关遗传损伤的发生或后果？3)McM2缺陷在多大程度上反映了处于增殖应激下的细胞中发生的变化？提出了四个目标。第一个将定义效果 对肿瘤中发生的遗传损伤的性质和部位进行不同的遗传背景研究，以解决导致肿瘤抑制的特定机制和基因。第二个将在全基因组范围内定义复制的启动最受影响的位置 不同遗传背景的MCM2缺乏症。第三个将测量在肿瘤形成之前由于胸腺组织中Mcm2缺乏而发生遗传损害的位置，以允许复制效率和遗传损害之间的直接关联，而不需要对肿瘤进行任何额外的选择。第四个将确定在McM2缺乏条件下观察到的变化在多大程度上也在增殖应激条件下出现。这些研究将为预测导致特定疾病的遗传损害的易感性和减轻这种易感性的后果提供信息。

DESCRIPTION (provided by applicant): Accurate replication of DNA during cellular division is central to maintaining genome integrity where the consequence inaccurate replication is genomic damage leading to cancer and other age related dysfunction. These studies will inform approaches to strengthening mechanism required for accurate DNA replication and predicting the locations where damage is likely to occur when these mechanisms fail. The project is based on a transgenic mouse line developed by this laboratory that carries a subtle mutation resulting in insufficient expression of a core component of the replication machinery, Mcm2, which is proving to be extremely valuable in understanding the consequences of deficient replication origin licensing. These mice are remarkably cancer prone and exhibit complete penetrance of thymic lymphoblastic lymphoma on the 129Sv genetic background on which it was constructed. Studies supported by this project have implicated inefficient initiation of replication from primar origins and, to a greater extent, dormant (backup) origins under conditions of replication fork stalling and collapse as a major reason for this susceptibility. Additionally, the genetic damage in the resulting tumors is in the form of short deletions that markedly facilitate identification o tumor suppressor genes. Further, these studies have shown that some strains of mice differ in their ability to manage the consequences of Mcm2 deficiency as evidenced by reduced tumorigenesis. The proposed studies address three key issues: 1) To what degree does sequence specificity, either directly or as a consequence of epigenetic modification, in the sites at which DNA replication initiates influence the locations at which replication related genetic damage is likely to arise in Mcm2 deficient mice? 2) Do genetic factors that mitigate the disease phenotypes resulting from Mcm2 deficiency on alternative genetic backgrounds function through an effect on the efficiency of origin licensing at specific locations or are there alternative mechanisms that suppress the occurrence or consequences of replication related genetic damage in these strains? 3) To what extent does Mcm2 deficiency reflect changes that occur in cells that are under proliferative stress? Four aims are proposed. The first will define the effect of different genetic backgrounds on the properties and sites of genetic damage that occurs in tumors to address specific mechanisms and genes responsible for tumor suppression. The second will define genome wide the locations at which initiation of replication is most affected by Mcm2 deficiency on different genetic backgrounds. The third will measure the sites at which genetic damage occurs due to Mcm2 deficiency in thymic tissue prior to the formation of tumors to allow correlation between replication efficiency and genetic damage directly, in the absence of any additional selection for tumorigensis. The fourth will determine the extent to which changes observed under conditions of Mcm2 deficiency are also seen under conditions of proliferative stress. These studies will inform approaches to predicting predisposition to genetic damage resulting in specific disease and mitigating the consequences of such predisposition.