Cell Proliferation in Genome and Tissue Integrity

描述(申请人提供)：由于衰老导致适应性反应的退化和失败，导致健康恶化、发病率增加和疾病易感性。现在有相当多的人支持这样的观点，即DNA累积损伤是这种损失的很大一部分原因。拟议研究的更大目标是确定遗传损害积累的主要驱动因素以及减轻这些损害的机制。这项拟议的工作将对细胞复制在遗传损伤积累中的作用进行关键测试。此外，目前工作的一个关键重点是细胞在复制分叉停滞后招募休眠起源的能力，使细胞能够从这一事件中恢复，而不需要使用产生DNA断裂的修复机制和遗传损伤的可能性。通过这种方式，休眠的起源充当了对 复制相关的基因损伤。使用两个独特的小鼠模型，我们试图解决的关键问题是：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)通过休眠起源影响复制拯救效率的分子机制是什么？利用可以诱导体内细胞增殖抑制的小鼠模型，以及初步研究表明神经干细胞在体内经历的细胞分裂比同龄对照动物少得多的情况下，将确定增殖减少对遗传损伤累积水平的影响。阵列CGH将作为这些小鼠克隆神经球遗传损伤累积的全基因组读数。这些研究有望为细胞增殖在体内DNA损伤积累中的作用提供一个明确的测试。第二种小鼠模型由于复制前复合体的关键成分McM2的表达不足而导致休眠的起源功能受损，该模型将用于确定减少起源许可对基因组稳定性的影响，并筛选可以改善起源许可功能的机制。对McM2缺陷小鼠肿瘤的初步研究表明，休眠起源功能失效的一个后果是更频繁的较短长度的间质染色体缺失，这可能是一个特征。这种可能性将被测试，并与DNA纤维分析一起使用，以测试可能影响功能的候选基因的作用。来自McM2基因缺陷小鼠的细胞还将被用于筛选汇集的shRNA文库，以根据合成致死性或功能挽救来筛选可能影响休眠起源功能的基因。这些研究有望提高我们对休眠起源功能的理解，并提供加强这一活动的方法。这些研究的一个潜在结果是，在没有细胞增殖减少的负面后果的情况下，确定了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.