CELL BIOLOGY OF OXIDATIVE DNA DAMAGE AND REPAIR

描述(申请人的描述)：由 活性氧类与衰老和年龄相关 疾病，以及多种形式的人类癌症。8-氧鸟嘌呤是一种病变 它已被用作氧化DNA损伤的标志。8-氧鸟嘌呤已经被 在体内和体外都有致突变性。最近，猫1，一只小鼠8- 氧鸟嘌呤-DNA修复酶的克隆及其在转基因中的高效表达 动物。尽管已经积累了大量关于衬底的信息 Mogg1及其异构体的特异性和修复机制 关于它们的生化特性的信息很少，在 正常和正常生长的细胞的细胞周期和亚细胞分布 氧化应激状态。同样，关于以下方面的信息很少 Ogg1在细胞核内的分布及其与人类免疫缺陷的关系 染色质和细胞核的结构成分。我们建议解决以下问题 通过提出针对纯化的单特异性抗体来解决这些问题 B A野生型重组MOGG1。我们将使用这些 确定mogg1和mogg1亚细胞定位的研究进展 酶衍生的核和细胞质池的生化特征 来自哺乳动物的组织培养细胞以及野生型和 转基因小鼠过表达ogg1。我们还将描述归纳的特征 通过确定营养缺乏细胞中的氧化DNA损伤 8-氧鸟嘌呤水平与ogg1的合成和热休克有关 蛋白质，并评估ogg1对细胞周期的依赖调节。最后， 我们将3-氧鸟嘌呤核“热点”与ogg1、染色质和 组织培养细胞中细胞核的结构蛋白成分和 野生型和转基因小鼠的组织过表达mogg1。

DESCRIPTION (Applicant's Description): Oxidative DNA damage induced by reactive oxygen species has been associated with aging and age-elated diseases, as well as several forms of human cancer. 8-Oxoguanine is a lesion that has been used as a marker for oxidative DNA damage. 8-Oxoguanine has been shown to be mutagenic in vivo and in vitro. Recently, mogg1, a murine 8- oxoguanine-DNA repair enzyme was cloned and over expressed in transgenic animals. Although extensive information has been accumulated on the substrate specificity and the repair mechanism of mogg1 and its isoforms, there is little information regarding their biochemical properties, regulation during the cell cycle and subcellular distribution in cells growing under normal and oxidative stress conditions. Similarly, there is little information regarding the intranuclear distribution of ogg1 and its molecular relationships with chromatin and structural components of the nucleus. We propose to address these questions by raising monospecific antibodies directed against purified b a c terially-expressed wild-type recombinant mogg1. We will use these a n t i b odies to determine the subcellular localization of mogg1 and biochemically characterize nuclear and cytoplasmic pools of the enzyme derived from mammalian tissue culture cells as well as the liver of wild-type and transgenic mice over expressing ogg1. We will also characterize the induction of oxidative DNA damage in nutrient deprived cells by determining whether levels of 8-oxoguanine correlate with the synthesis of ogg1 and heat shock proteins and evaluating the cell cycle-dependent regulation of ogg1. Finally, we will colocalize 3-oxoguanine nuclear "hot spots" with ogg1, chromatin and structural protein components of the nucleus in tissue culture cells and tissues from wild-type and transgenic mice over expressing mogg1.