Chemistry and Biology of Oxidized Purine Lesions in DNA

描述（由申请人提供）：DNA修复受损与人类癌症发展之间的致病作用已经确立。由于内源性氧化剂、感染和损伤的炎症反应以及暴露于氧化还原活性环境毒素，DNA碱基受到鸟嘌呤和其他碱基化学修饰的氧化应激。虽然很多注意力都集中在氧化鸟嘌呤病变8-氧鸟嘌呤（OG）导致的G到T突变上，但本项目研究了OG进一步氧化导致的高氧化鸟嘌呤病变。这些病变构成了一个高度诱变的海因结构家族，包括螺酰亚胺二海因（Sp）和胍酰海因（Gh），除了G到T突变外，它们似乎还导致G到C突变。在本工作中，我们将对这类化合物的相关成员进行研究，进一步探索氧化途径、产物结构和活性与DNA加工酶的关系，包括碱基切除修复和核苷酸切除修复机制。待研究的新结构包括氧化鸟苷的胺加合物。提出了新的途径，可能解释氧化应激致突变谱中的其他观察结果，包括双链串联病变的形成。这项工作的具体目标是：(1)通过使用合成含病变的寡脱氧核苷酸、质谱和x射线晶体学，解决围绕双链DNA中海因蛋白病变形成的结构问题；(2)通过体外和体内生化分析，特别关注nei样（hNEIL1）和Fpg糖基化酶的作用，了解碱基切除修复与核苷酸切除修复在海因蛋白病变中的作用；(3)开发创新的检测方法，通过生成DNA损伤适配体和荧光读数来检测细胞裂解物中的氢妥英蛋白产物。

DESCRIPTION (provided by applicant): A causative role has been established between compromised DNA repair and the development of human cancers. DNA bases are subject to oxidative stress undergoing chemical modification of guanine and other bases as a result of endogenous oxidants, inflammatory responses to infection and injury, and exposure to redox-active environmental toxins. While much attention has been focused on G to T mutations as a result of the oxidized guanine lesion 8-oxoguanine (OG), this project investigates the hyperoxidized guanine lesions resulting from further oxidation of OG. These lesions constitute a family of highly mutagenic hydantoin structures including spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) that appear to cause G to C in addition to G to T mutations. In the present work, related members of this class of compounds will be studied to further explore the relationship between oxidation pathway, product structure, and activity with DNA processing enzymes including base excision repair and nucleotide excision repair mechanisms. The new structures to be studied include amine adducts of oxidized guanosine. Novel pathways are proposed that might explain additional observations in the mutagenic spectrum of oxidative stress, including the formation of double-stranded tandem lesions. The specific aims of this work are to: (1) resolve structural questions surrounding the formation of hydantoin lesions in duplex DNA through the use of synthetic lesion-containing oligodeoxynucleotides, mass spectrometry, and x-ray crystallography, (2) understand the roles of base excision repair vs. nucleotide excision repair for hydantoin lesions via in vitro and in vivo biochemical assays with a particular focus on the role of the Nei-like (hNEIL1) and Fpg glycosylases, and (3) develop innovative assays to detect hydantoin products in cell lysates by generating aptamers to DNA lesions coupled to fluorescent readouts. PUBLIC HEALTH RELEVANCE: Oxidative stress plays a significant role in the damaging DNA creating lesions that underlie cancer, aging, neurological and cardiac disorders. This project will help define how specific oxidized DNA bases and base adducts lead to mutations related to cancer. In addition, methods will be developed to monitor the formation and excision of these damaged bases from DNA, revealing new information about the relationship between DNA damage and cancer.