Ring-Opened and Tandem DNA Damages

开环和串联 DNA 损伤

• 批准号: 7105322
• 负责人: Ashis K Basu
• 金额: $ 31.64万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-10 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7105322
• 关键词: DNA damage; DNA repair; adduct; biotechnology; cell line; circular dichroism; crosslink; functional /structural genomics; gene deletion mutation; ionizing radiation; mutagen testing; neoplasm /cancer genetics; neoplastic process; nuclear magnetic resonance spectroscopy; nucleic acid chemical synthesis; nucleic acid denaturation; oligonucleotides; oxidative stress; plasmids; transfection /expression vector

DESCRIPTION (provided by applicant): We hypothesize that a group of oxidative DNA damages, including 8-oxoguanine, formamidopyrimidines and tandem lesions, play important roles in the etiology of human cancer via the mutagenic and genotoxic effects of these lesions in certain important sequences of cancer genes. In this application, we propose to study the biological effects of a subset of these lesions. We have three specific aims. In aim 1, synthesis and characterization of oligonucleotides containing tandem base lesions in DNA will be carried out. Tandem lesions formed by ionizing radiation, transition metal/H2O2, or other radical- mediated processes include abasic site adjacent to 8-oxoguanine, S- and R-8,5'-cyclo-2'- deoxyguanosine, and intrastrand vicinal guanine-thymine cross-link. We shall determine the structural effects of these damages in DNA by thermal melting, circular dichroism, and NMR. In aim 2, single stranded plasmid or viral vectors containing these lesions will be constructed. Mutagenicity and repair studies of the site-specific lesions will be carried out in Escherichia coli and in mammalian cells. For example, we shall determine how mutagenicity of 8-oxoguanine might be influenced when it is present as part of a tandem DNA damage. We shall investigate if Fapy.dG, the guanine-thymine vicinal cross- link, S- and R-8, 5'-cyclo-2'-deoxyguanosine are mutagenic, genotoxic, or both in mammalian cells. In aim 3, we shall construct duplex plasmid vectors with these lesions, which will be used to examine the extent of lesion bypass by extracts of normal and repair- or replication-altered mammalian cells (e.g., isogenic mouse embryonic fibroblasts and cells that express modulated levels of DNA polymerases encoded by REV1, REV3, RAD18, etc. genes). We hope to contribute toward a deeper insight of the effects of oxidative DNA damages.

描述（由申请人提供）：我们假设一组氧化性DNA损伤，包括8-氧化氨基，甲氨基吡啶和串联病变，通过这些病变在某些重要的癌症基因的某些重要序列中的诱变和遗传毒性作用在人类癌症的病因中起重要作用。在此应用中，我们建议研究这些病变子集的生物学作用。我们有三个具体的目标。在AIM 1中，将执行含有串联碱病变的寡核苷酸的合成和表征。通过电离辐射，过渡金属/H2O2或其他自由基介导的过程形成的串联病变包括邻近8-氧气的可羟氨酸，S-和R-8,5,5'-Cyclo-2'-脱氧鸟苷，以及内氨酸情况豚鼠 - 鸟嘌呤 - 胰鸟嘌呤 - 胰岛素 - 胰岛素交叉链接。我们将通过热熔化，圆形二色性和NMR来确定DNA中这些损伤的结构效应。在AIM 2中，将构建包含这些病变的单链质粒或病毒向量。位点特异性病变的诱变性和修复研究将在大肠杆菌和哺乳动物细胞中进行。例如，我们将确定8-氧气的诱变性在存在作为串联DNA损伤的一部分时可能会受到如何影响。我们将研究Fapy.dg，鸟嘌呤 - 胸腺胸骨横链连接，S-8和R-8，5'-Cyclo-2'-脱氧鸟苷是诱变，遗传毒性或在哺乳动物细胞中都是诱变的。在AIM 3中，我们将使用这些病变构建双工质粒载体，该损伤将用于检查正常和修复或修复或复制的哺乳动物细胞的病变旁路的程度（例如，等源性小鼠胚胎成纤维细胞和细胞，这些细胞和细胞表达了由Rev1，Rev1，Rev1，Rev1，Rev1，Rev1，Rev1，Rev1，Rev1，Rev3，Rad，Rad，Rad，Rev1，Rev1，Rev1，Rev1，rad18等。我们希望有助于更深入地了解氧化DNA损伤的影响。