Replication Licensing in Genome Stability, Cancer and Aging

描述（由申请人提供）：细胞分裂期间DNA的准确复制对于维持基因组完整性至关重要，其中不准确复制的后果是基因组损伤，导致癌症和其他年龄相关功能障碍。这些研究将为加强精确DNA复制所需的机制以及预测这些机制失效时可能发生损伤的位置提供信息。该项目是基于该实验室开发的转基因小鼠品系，该品系携带导致复制机制核心成分Mcm2表达不足的微小突变，这被证明在理解复制起点许可缺陷的后果方面极具价值。这些小鼠是显着的癌症倾向，并表现出胸腺淋巴母细胞淋巴瘤的129Sv遗传背景上，它是构建的完全转移率。由该项目支持的研究表明，在复制叉停滞和崩溃的条件下，从主要起源和在更大程度上休眠（备份）起源的复制启动效率低下是这种易感性的主要原因。此外，所产生的肿瘤中的遗传损伤是以短缺失的形式，这显著地促进了肿瘤抑制基因的鉴定。此外，这些研究表明，一些品系的小鼠在处理Mcm2缺乏的后果方面的能力不同，如肿瘤发生减少所证明的。拟议的研究解决了三个关键问题：1）在何种程度上序列特异性，直接或作为表观遗传修饰的结果，在DNA复制启动的位置影响复制相关的遗传损伤的位置可能会出现在Mcm2缺陷小鼠？2)是否遗传因素，减轻疾病表型造成的Mcm2缺陷的替代遗传背景的功能，通过对效率的原产地许可证在特定的位置或有替代机制，抑制发生或后果的复制相关的遗传损伤，在这些菌株？3)Mcm2缺陷在多大程度上反映了细胞在增殖应激下发生的变化？提出了四个目标。第一个将定义效果 不同遗传背景对肿瘤中发生的遗传损伤的性质和位点的影响，以解决肿瘤抑制的特定机制和基因。第二个将定义全基因组范围内的位置，在那里复制的起始是最受影响的 不同遗传背景下的Mcm2缺陷。第三个将测量肿瘤形成之前由于胸腺组织中Mcm 2缺陷而发生遗传损伤的位点，以在没有任何额外的致瘤选择的情况下，允许复制效率和遗传损伤之间直接相关。第四项将确定在Mcm2缺乏条件下观察到的变化在增殖应激条件下也能观察到的程度。这些研究将为预测导致特定疾病的遗传损伤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.