Mechanisms of selective excision and oxidative repair of alkylated DNA

描述（由申请人提供）：n3 -甲基腺嘌呤（3mA）和1，n6 -乙烯腺嘌呤（eA）是暴露于环境遗传毒性物质、细胞代谢物和抗癌药物后产生的两种DNA碱基修饰。3mA病变具有高度的细胞毒性，因为它们抑制聚合酶的DNA合成，这种细胞毒性是在癌症化疗中使用烷基化剂的基本原理。eA与慢性炎症有关，具有高度诱变性，可导致基因组不稳定和癌症。两种不同的，部分冗余的酶活性已经进化为这两种特定的病变：i) DNA双加氧酶的氧化去甲基化和ii) DNA糖基化酶的碱基切除修复。这些酶对3mA和eA的特异性和催化作用的确切决定因素尚不清楚。我们试图通过定向进化和结构生物学方法的独特整合来填补这一关键的知识空白，以获得对人类ABH2双加氧酶（Aim 1）和酵母MAG 3mA糖基酶家族（Aim 2）的3mA和eA选择和催化的全面机制理解。这项工作利用了在两种修复系统之间观察到的趋同进化，并基于我们已经确定的具有保护细胞免受3mA毒性的ABH2突变体的初步结果。我们将验证ABH2修复3mA的假设，与其他已知底物的修复不同，它涉及碱基切除修复的切除和进一步加工。我们对每个目标的一般方法是i)在选择性烷基化压力下使用定向进化识别对底物识别重要的残基，ii)确定ABH2和MAG蛋白与3mA-和eA- dna复合物的晶体结构，以及iii)测试单个残基对3mA和eA特异性和修复的贡献。这些研究将为这些酶如何决定细胞毒性和致突变病变的命运提供新的见解，以实现特定的修复途径。此外，在Aim 1中，我们利用小鼠红细胞白血病（MEL）细胞组织培养模型探讨了ABH2突变体在甲基化药物治疗癌症中的翻译意义。我们的研究至少有三个直接的临床意义。首先，乙烯- dna加合物可能在与慢性炎症相关的癌症病因学中发挥作用，因此eA修复的结果可能为确定慢性炎症患者的癌症风险提供新的方法。其次，我们的3ma保护ABH2突变体对理解肿瘤中甲基化药物治疗耐药的起源以及设计涉及骨髓保护的新化疗方法具有直接意义。第三，我们对3mA糖基酶修复的结构-功能研究是设计小分子抑制剂以增强甲基化剂细胞毒性的必要的第一步。

DESCRIPTION (provided by applicant): N3-methyladenine (3mA) and 1,N6-ethenoadenine (eA) are two DNA base modifications produced from exposure to environmental genotoxic agents, cellular metabolites, and anti-cancer drugs. 3mA lesions are highly cytotoxic owing to their inhibition of DNA synthesis by polymerases, and this cytotoxicity is a rationale for the use of alkylating agents in cancer chemotherapy. eA, which is associated with chronic inflammatory conditions, is highly mutagenic and can lead to genomic instability and cancer. Two different, partially redundant enzymatic activities have evolved for these two specific lesions: i) oxidative demethylation by DNA dioxygenases and ii) base excision repair by DNA glycosylases. The precise determinants for the specificity and catalysis of these enzymes toward 3mA and eA remain unclear. We seek to fill this critical gap in knowledge by a unique integration of directed evolution and structural biology methods in order to obtain a comprehensive mechanistic understanding of 3mA and eA selection and catalysis by human ABH2 dioxygenase (Aim 1) and the yeast family of MAG 3mA glycosylases (Aim 2). This work capitalizes on the convergent evolution observed between the two repair systems, and is based on our preliminary results that have identified ABH2 mutants with the capacity to protect cells from 3mA toxicity. We will test the hypothesis that ABH2 repair of 3mA, unlike that of other known substrates, involves excision and further processing by base excision repair. Our general approach for each aim is to i) identify residues important for substrate discrimination using directed evolution under selective alkylation pressure, ii) determine crystal structures of ABH2 and MAG proteins in complex with 3mA- and eA-DNA, and iii) test the contribution of individual residues toward 3mA and eA specificity and repair. These studies will provide novel insight into how these enzymes determine the fate of cytotoxic and mutagenic lesions toward a particular repair pathway. In addition, in Aim 1 we probe the translational implications of our ABH2 mutants for cancer treatment with methylating agents using a mouse erythroleukemia (MEL) cell tissue culture model. Our studies have at least three direct clinical implications. First, etheno-DNA adducts likely play a role in the etiology of cancer associated with chronic inflammation, and thus results on eA repair may provide new ways to determine the risk of cancer in patients suffering from chronic inflammatory conditions. Second, our 3mA-protecting ABH2 mutants have direct implications for understanding the origins of resistance to therapy with methylating agents in tumors and for the design of new chemotherapeutic approaches involving bone marrow protection. Third, our structure-function studies on 3mA glycosylase repair are a necessary first step for the design of small molecule inhibitors as a way to enhance the cytotoxicity of methylating agents.