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.
The larger objective of the present studies is to improve our understanding of the mechanisms underlying the substantial declines of function and adaptive capacities in aged individuals. A unique set of transgenic reagents and reporter lines will be used to probe the role of cell proliferation and DNA replication in the genetic damage accumulation that underlies much of this decline and to define mechanisms by which modulating replication origin function may mitigate the accumulation of this damage. This work will inform approaches to reducing genetic damage accumulation in the absence of the negative consequences of reduced cellular proliferation.
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