Mechanisms of selective excision and oxidative repair of alkylated DNA

烷基化DNA的选择性切除和氧化修复机制

基本信息

批准号：
8878258
负责人：
Manel Camps
金额：
$ 33.72万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA CRUZ
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-25 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8878258
关键词：
A-Form DNA Acute Erythroblastic Leukemia Address Adenine Alkylating Agents Alkylation Antineoplastic Agents Base Excision Repairs Biochemical Bone Marrow Cancer Etiology Catalysis Cell Death Cells Chemotherapy-Oncologic Procedure Chronic Clinical Complex DNA DNA Adducts DNA Alkylation DNA Damage DNA Repair Enzymes DNA Synthesis Inhibition DNA biosynthesis DNA glycosylase Data Dealkylation Dioxygenases Discrimination Enzymes Escherichia coli Evolution Excision Exhibits Exposure to Family Fission Yeast Foundations Genetic Genome Stability Genomic Instability Goals Health Human Individual Inflammation Inflammatory Knowledge Laboratories Lead Lesion Malignant Neoplasms Mammalian Cell Metabolism Methods Methylation Modeling Modification Molecular Mus Mutagens Myelin Associated Glycoprotein Normal Cell Nucleotides Oligonucleotides Organism Orthologous Gene Pathway interactions Patients Phylogenetic Analysis Play Polymerase Process Reactive Oxygen Species Resistance Role Specificity Structure Substrate Specificity System Testing Therapeutic Toxic Environmental Substances Toxic effect Work Yeasts adduct base cancer risk cancer therapy chemotherapy comparative cytotoxic cytotoxicity demethylation design directed evolution human DNA improved inhibitor/antagonist insight mutant novel oxidant stress pressure repaired small molecule structural biology therapy resistant tissue/cell culture tool tumor

项目摘要

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.

描述（由申请人提供）：N3-甲基丹宁（3mA）和1，N6-乙烯二烯（EA）是通过暴露于环境遗传毒性剂，细胞代谢产物和抗癌药物而产生的两种DNA碱基修饰。由于聚合酶对DNA合成的抑制作用，3MA病变具有高度细胞毒性，并且该细胞毒性是在癌症化学疗法中使用烷基化剂的理由。与慢性炎症条件相关的EA是高度诱变的，可以导致基因组不稳定性和癌症。这两个特定的病变已经进化了两种不同的部分冗余酶活性：i）通过DNA二加氧酶氧化脱甲基化，ii）II）通过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差距，以便获得对人类ABH2二氧酶的3MA和EA选择和催化（AIM 1）和MAG 3MA糖基酶的酵母菌家族的全面机械理解（AIM 1）（AIM 2）。这项工作利用了两个修复系统之间观察到的收敛进化，并基于我们的初步结果，这些结果已经确定了具有保护细胞免受3MA毒性的能力的ABH2突变体。我们将检验以下假设：与其他已知底物不同，ABH2修复3MA涉及切除和通过基础切除修复进行进一步处理。我们针对每个目标的一般方法是i）确定在选择性烷基化压力下使用定向进化的底物歧视的残基，ii）确定与3mA-和EA-DNA复合物中ABH2和MAG蛋白的晶体结构，以及III）测试单个残基对3MA和EA的特异性和EA的特异性和修复的贡献。这些研究将提供有关这些酶如何确定细胞毒性和诱变病变对特定修复途径的命运的新见解。此外，在目标1中，我们使用小鼠红血病（MEL）细胞组织培养模型探测了ABH2突变体对用甲基化剂进行癌症治疗的翻译意义。我们的研究至少具有三个直接临床意义。首先，埃塞诺-DNA加合物可能在与慢性炎症有关的癌症病因中起作用，因此EA修复的结果可能会提供新的方法来确定患有慢性炎症状况的患者的癌症风险。其次，我们的3MA保护ABH2突变体对理解肿瘤中甲基化剂对治疗的抗药性的起源以及涉及骨髓保护的新化学治疗方法的设计。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的必要第一步，以增强甲基化剂的细胞毒性。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Non-productive DNA damage binding by DNA glycosylase-like protein Mag2 from Schizosaccharomyces pombe.

DOI：
10.1016/j.dnarep.2012.12.001
发表时间：
2013-03-01
期刊：
DNA REPAIR
影响因子：
3.8
作者：
Adhikary, Suraj;Cato, Marilyn C.;McGary, Kriston L.;Rokas, Antonis;Eichman, Brandt F.
通讯作者：
Eichman, Brandt F.

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli.