CELL BIOLOGY OF OXIDATIVE DNA DAMAGE AND REPAIR

描述（申请人描述）： 氧化损伤 活性氧与衰老和老化相关 疾病，以及几种形式的人类癌症。8-氧鸟嘌呤是一种 它被用作氧化性DNA损伤的标记。8-氧代鸟嘌呤已被 在体内和体外均显示出致突变性。 最近，一只8- 氧代鸟嘌呤-DNA修复酶被克隆并在转基因中过表达， 动物 尽管已经积累了大量关于基底的信息 特异性和修复机制的mogg 1及其亚型，有 关于它们的生化特性， 在正常生长条件下生长的细胞的细胞周期和亚细胞分布， 氧化应激条件。同样，关于 ogg 1在核内的分布及其与 染色质和细胞核的结构成分。我们建议解决 这些问题通过产生针对纯化的 B体外表达的野生型重组mogg 1。 我们将会用这些 本B方法测定了mogg 1的亚细胞定位， 生物化学表征酶衍生物的细胞核和细胞质池 来自哺乳动物组织培养细胞以及野生型和 过表达ogg 1的转基因小鼠。我们还将描述归纳 在营养缺乏的细胞中，通过确定是否 8-氧代鸟嘌呤的水平与ogg 1的合成和热休克相关 蛋白质和评估ogg 1的细胞周期依赖性调节。最后， 我们将3-氧代鸟嘌呤核“热点”与ogg 1、染色质和 组织培养细胞中细胞核的结构蛋白组分， 来自野生型和过表达MOGG 1的转基因小鼠的组织。

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.