Cell Proliferation in Genome and Tissue Integrity

描述（由申请方提供）：由于衰老导致的适应性反应的退化和失效导致健康恶化、发病率增加和对疾病的易感性。现在有大量的证据支持这种观点，即累积的DNA损伤是这种损失的重要部分。拟议研究的更大目标是确定遗传损伤积累的主要驱动因素以及减轻这些因素的机制。这项工作将为细胞复制在遗传损伤积累中的作用提供一个关键的测试。此外，目前工作的一个关键重点是细胞在复制叉停滞后招募休眠起点的能力，允许细胞从这一事件中恢复，而不需要采用产生DNA断裂的修复机制和遗传损伤的可能性。以这种方式，休眠的起源作为第一道防线， 复制相关的遗传损伤。使用两种独特的小鼠模型，我们寻求解决的关键问题是：1）长期抑制细胞增殖对体内遗传损伤积累的影响是什么？2)什么样的分子机制影响了通过休眠起源的复制拯救的效率？ 使用小鼠模型，其中可以诱导体内细胞增殖的抑制，并且其中初步研究表明神经干细胞在体内经历的细胞分裂比在同等年龄的对照动物中发生的细胞分裂少得多，将确定增殖减少对遗传损伤积累水平的影响。阵列CGH将作为来自这些小鼠的克隆神经球的遗传损伤累积的全基因组读出进行。预期这些研究将提供细胞增殖对体内DNA损伤积累作用的确定性测试。 第二个小鼠模型，其中休眠的起源功能受到损害，由于缺乏表达的一个关键组成部分的复制前复合物，Mcm2，将被用来定义减少的起源许可对基因组稳定性的后果，并筛选机制，可以提高起源许可功能。从Mcm2缺陷小鼠产生的肿瘤的初步研究表明，休眠起源功能失败的一个后果，这可能是一个签名，是更频繁的间质染色体缺失的长度较短。这种可能性将被测试，并与DNA纤维分析结合使用，以测试可能影响功能的候选基因的作用。来自Mcm2缺陷型小鼠的细胞将另外用于基于合成致死性或功能拯救来筛选可影响休眠起源功能的基因的汇集的shRNA文库。预计这些研究将提高我们对休眠起源功能的理解，并为增强这种活动的方法提供信息。这些研究的一个潜在结果是确定在没有细胞增殖减少的负面后果的情况下减少遗传损伤积累的方法。

DESCRIPTION (provided by applicant): Degradation and failure of adaptive responses due to aging result in health deterioration, increased morbidity and susceptibility to disease. There is now substantial support for the idea that accumulated DNA damage underlies a significant portion of this loss. The larger objective of the proposed studies is to identify the primary drives of genetic damage accumulation and mechanisms by which they can be mitigated. The proposed work will provide a critical test of the role of cellular replication in genetic damage accumulation. Additionally, a key focus of the present work is on the ability of cells to recruit dormant origins following replication fork stalling, allowing cells to recover from this event without the necessity of employing repair mechanisms that generate DNA breaks and the possibility of genetic damage. In this way dormant origins serve as a first line of defense against replication related genetic damage. Using two unique mouse models, the key questions we seek to address are: 1) What is the effect of long term suppression of cell proliferation on accumulation of genetic damage in vivo? 2) What molecular mechanisms influence the efficiency of replication rescue through dormant origins? Using a mouse model in which suppression of cell proliferation in vivo can be induced, and where preliminary studies demonstrate that neural stem cells have undergone far fewer cell divisions in vivo than occur in control animals of equivalent age, the effect of reduced proliferation on the level of genetic damage accumulation will be determined. Array CGH will be performed as a genome-wide readout of genetic damage accumulation from clonal neurospheres from these mice. These studies are anticipated to provide a definitive test of the role of cell proliferation on DNA damag accumulation in vivo. A second mouse model in which dormant origin function is compromised due to deficient expression of a key component of the prereplicative complex, Mcm2, will be used to define the consequences of reduced origin licensing on genome stability and to screen for mechanisms that can improve origin licensing function. Preliminary studies from tumors arising on Mcm2 deficient mice suggest that one consequence of the failure of dormant origin function, which may serve as a signature, is more frequent interstitial chromosomal deletions of shorter length. This possibility will be tested and used in conjunction with DNA fiber analysis to test the role of candidate genes that may affect function. Cells from Mcm2 deficient mice will additionally be used to screen pooled shRNA libraries for genes that can affect dormant origin function based on synthetic lethality or rescue of function. These studies are anticipated to improve our understanding of dormant origin function and to inform approaches to enhancing this activity. A potential outcome of these studies is identification of approaches to reduced genetic damage accumulation in the absence of the negative consequence of reduced cellular proliferation.