OXIDATIVE STRESS,REPAIR OF OXIDIZED GUANINES IN AGED CELLS

活性氧物种(ROS)通过改变维持细胞内稳态的细胞信号级联反应而导致组织功能下降，并导致包括DNA在内的细胞成分的损伤，从而与病因和衰老有关。8-氧鸟嘌呤(8-oxoG)和开环嘌呤(FapyG)是ROS诱导的两种丰富的突变和毒性碱基损伤，主要由8-oxoG-DNA糖基酶(OGGI)修复，其剪接变异体OGGI-1a和OGG1-2a分别针对细胞核和线粒体(MR)。在OGG 14-鼠细胞中，这些损伤同时聚集在核和线粒体DNA中，与增强的突变和自发性董腺癌有关。同时，8-oxoG/FapyG在衰老过程中缺乏有效的核输入和基因组中损伤的积累支持8-oxoG/FapyG的病因学参与。矛盾的是，8-oxoG/FapyG在细胞核和线粒体DNA中随年龄的增加而积累，而OGG1总活性没有下降。这种差异可以用我们的结果解释，我们的结果显示，在衰老的细胞中，Ogg1-2a的mt进口很少，因此很大一部分酶仍然与mt外膜结合。此外，ROS诱导的OGG1-1a在衰老细胞中的核积聚被推迟。 该项目的中心假设是，毒性和诱变性氧化DNA损伤的积累是由于衰老细胞维持细胞核和线粒体中正常Ogg 1水平的能力降低，从而导致组织功能下降。利用各种细胞、分子和转基因方法，并与P1和P2合作，我们将检验我们的假设，目的如下：1)探索年龄相关性缺陷的机制 目的：2)验证OGG1核积累延迟与其在OGG1-1a中确定的共价修饰(例如乙酰化)有关的初步观察；以及3)确认8-oxoG/FapyG在衰老细胞mtDNA中的积累确实是由于OGG1-2a低效靶向受ROS影响的mt基质而导致修复减少所致。这些研究将对衰老过程中主要诱变性和毒性损伤的积累提供重要的线索。长期目标是开发干预策略，以改善与年龄相关的细胞核和腐烂的突变DNA损伤修复能力的下降，从而延缓线粒体和细胞功能的下降。

Reactive oxygen species (ROS) are implicated in the etiology &aging by causing a decline in tissue functions due to altered cellular signaling cascades that maintain cellular homeostasis, and by inducing damage to cellular components, including DNA. 8-Oxoguanine (8-oxoG), and ring-opened purine (FapyG), the two abundant mutagenic and toxic base lesions induced by ROS, are repaired primarily by 8-oxoG-DNA glycosylase (OGGI), with spliced variants, OGGI-1 a and OGG1-2a, targeted to nucleus and mitochondria (mr), respectively. In ogg 14-mouse cells, these lesions accumulate in both nuclear and mtDNA, associated with enhanced mutagenesis, and spontaneous tung carcinoma. At the same time, lack of efficient nuclear import of OGG 1-1 a and accumulation of damage in the genome of senescence-accelerated mice support the etiologic involvement of 8-oxoG/FapyG in aging processes. Paradoxically, age-dependent accumulation of 8-oxoG/FapyG in nuclear and mtDNA occurs without a decline in total OGG1 activity. This discrepancy could be explained by our results showing poor mt import of OGG 1-2a in senescent cells so that a significant fraction of the enzyme remains bound to the outer mt membrane. Furthermore, nuclear accumulation of OGG1-1 a induced by ROS is delayed in the aged cells. The central hypothesis of this project is that toxic and mutagenic oxidative DNA lesions accumulate due to a decreased ability of aged cells to maintain normal levels of OGG 1 in the nucleus and mt thereby causing a decline in tissue functions. Using a variety of cellular, molecular and transgenic approaches, and in collaboration with P1 and P2, we will test our hypothesis with the following aims: 1) to explore the mechanism of age-dependent deficiency in repair of oxidative lesions after oxidative challenge; 2) to validate the preliminary observation that the delay in OGG1 nuclear accumulation is linked to its covalent modification (e.g., acetylation) identified in OGG1-la; and 3) to confirm that accumulation of 8-oxoG/FapyG in the mtDNA of aged cells is indeed due to reduced repair caused by inefficient targeting of OGG1-2a to the mt matrix, which is affected by ROS. These studies will shed significant light on the accumulation of the major mutagenic and toxic lesions in the aging process. The long-term objective is to develop intervention strategies to ameliorate an age-associated decrease in repair of mutagenic DNA lesions in the nucleus and rot, and thus to delay the decline in mitochondria and cellular functions.