REPAIR OF MUTAGENIC 8-OXOGUANINE IN MAMMALIAN GENOMES

8-氧鸟苷(8-oxoguanine，G*)是最重要的致突变和致癌DNA损伤，由电离辐射(IR)和其他环境因素诱发，或由氧化应激(通常被用作细胞氧化损伤的敏感标志物)内源性形成。G*是致突变的，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)途径进行，并通过G*-DNA糖基酶(OGG)去除G*而启动。人类主要的Ogg(Hogg-1)是在1997年克隆的。在人类细胞中缺乏OGG活性的早期证据现在似乎是由于G*特异结合蛋白(OGBP)的生理作用未知，Hogg-1与最近发现的线粒体OGG之间的关系也是如此。克隆的Hogg-1从G*C中移除G*，但不从G*A对中移除，这与后一反应将在DNA中形成G*时修复突变的原理一致。然而，G*也可以从核苷酸池中结合到DNA中，需要从新生的链中移除以防止突变。第二个Ogg(Ogg-2)从G*A对中去除G*，在抗原性上与Hogg-1不同，现在在人类细胞和酵母中都发现了它。我们对人类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是新生链特异性的，并利用错配修复系统。这个项目的主要目的是检验从模型中得出的假说，并阐明OGBP在G*的抗突变过程中的可能作用。该项目的具体目标是：(1)克隆Hogg-2并生产用于协作结构研究的重组OGG多肽；(2)检验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底物范围和底物识别的结构基础；(4)纯化和鉴定OGBP，以阐明其生物学作用；以及(5)研究线粒体和核OGG之间的关系。

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.