Chemistry and Biology of Oxidized Purine Lesions in DNA

描述(由申请人提供)：在受损的DNA修复和人类癌症的发展之间已经确立了因果关系。由于内源性氧化剂、对感染和损伤的炎症反应以及暴露在氧化还原活性环境毒素中，DNA碱基受到氧化应激，经历鸟嘌呤和其他碱基的化学修饰。由于8-氧鸟嘌呤(8-oxogguanine，OG)的氧化损伤，G到T突变引起了人们的极大关注，本项目研究了由于OG的进一步氧化而导致的高氧化的鸟嘌呤损伤。这些病变构成了一个高度突变的海因结构家族，包括螺亚氨基二海因(Sp)和胍基海因(Gh)，它们似乎除了导致G到T突变外，还可能导致G到C突变。在本工作中，将对这类化合物的相关成员进行研究，以进一步探索氧化途径、产物结构与DNA加工酶活性之间的关系，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，可以解释氧化应激突变谱中的其他观察结果，包括双链串联损伤的形成。这项工作的具体目的是：(1)通过使用合成的含有损伤的寡核苷酸、质谱学和X射线结晶学，解决围绕双链DNA中海因损伤形成的结构问题；(2)通过体外和体内生化分析，了解海因损伤的碱基切除修复与核苷酸切除修复的作用，尤其是Nei-like(HNEIL1)和FpG糖基酶的作用；以及(3)通过将适体与DNA损伤连接到荧光读数，来开发检测细胞裂解产物中的海因产物的创新方法。

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.