REPAIR OF MUTAGENIC 8-OXOGUANINE IN MAMMALIAN GENOMES

8-氧鸟嘌呤（8-oxoguanine, G*）可以说是最重要的致突变性和致癌性DNA损伤，它是由电离辐射（IR）和其他环境因子诱导的，或者是由于氧化应激内源性形成的（通常用作细胞氧化损伤的哨兵标记物）。G*具有诱变性，因为它在DNA合成过程中与A错配。DNA中G*的修复通过碱基切除修复（BER）途径发生，并通过G*-DNA糖基化酶（OGG）去除它而启动。主要的人类OGG （hOGG-1）于1997年被克隆。人类细胞中OGG活性的早期证据缺乏，现在似乎是由于G*特异性结合蛋白（OGBP）的生理作用未知，正如hOGG-1与最近发现的线粒体OGG之间的关系一样。克隆的hOGG-1将G*从G* C中移除，但没有从G* A对中移除，这与后一种反应将在DNA中形成G*时修复突变的原理一致。然而，G*也可以与核苷酸库中的DNA结合，需要从新生链中移除以防止突变。第二种OGG （OGG- 2）从G* A对中去除G*，从抗原性上不同于hOGG-1，目前已在人类细胞和酵母中发现。人类OGG-2的发现使我们提出了一种修复DNA中G*的新模型。我们的模型预测，OGG-2具有新生链特异性，并利用错配修复系统。该项目的主要目的是验证从该模型中得出的假设，并阐明OGBP在G*抗诱变加工中的可能作用。本项目的具体目标是：(1)克隆hOGG-2，制备重组OGG多肽，用于协同结构研究；(2)验证hOGG-2与错配修复和DNA复制复合体相互作用的假设；(3)比较hOGG-1和hOGG-2的底物范围和底物鉴别的结构基础；(4)对OGBP进行纯化和表征，阐明其生物学作用；(5)研究线粒体与细胞核ogg的关系。

8-oxoguanine (G*), arguably the most important mutagenic and carcinogenic DNA lesion, is induced by ionizing radiation (IR) and other environmental agents, or is formed endogenously due to oxidative stress (commonly used as the senitel marker for cellular oxidative damage). G* is mutagenic because of its mispairing with A during DNA synthesis. Repair of G* in DNA occurs via the base excision repair (BER) pathway, and is initiated with its removal by a G*-DNA glycosylase (OGG). The major human OGG (hOGG-1) was cloned in 1997. The lack of earlier evidence for OGG activity in human cells now appears to be due to a G*-specific binding protein (OGBP) whose physiological role is unknown, as is the relationship between the hOGG-1 and a recently identified mitochondrial OGG. The cloned hOGG-1 removes G* from an G* C, but not from an G* A pair, consistent with the rationale that the latter reaction will fix mutations when G* is formed in DNA. However, G* can also be incorporated into DNA from the nucleotide pool, requiring removal from the nascent strand to prevent mutation. A second OGG (OGG- 2), which removes G* from G* A pairs and is antigenically distinct from hOGG-1, has now been identified in both human cells and yeast. Our discovery of human OGG-2 has led us to propose a novel model for repair of G* in DNA. Our model predicts that OGG-2 is nascent-strand specific and utilizes the mismatch repair system. The primary objective of this project is to test the hypotheses derived from the model, and to elucidate the possible role of OGBP in the antimutagenic processing of G*. The specific aims of this project are: (1) To clone hOGG-2 and produce recombinant OGG polypeptides for collaborative structural studies; (2) to test the hypothesis that hOGG-2 interacts with mismatch repair and DNA replication complexes; (3) to compare the structural bases for the substrate range and substrate discrimination of hOGG-1 and hOGG-2; (4) to purify and characterize OGBP in order to elucidate its biological role; and (5) to investigate the relationship between the mitochondrial and nuclear OGGs.