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定了因果关系。由于内源性氧化剂、对感染和损伤的炎症反应以及暴露于氧化还原活性环境毒素，DNA碱基经受鸟嘌呤和其他碱基的化学修饰的氧化应激。虽然很多注意力都集中在G到T的突变作为氧化鸟嘌呤病变8-氧代鸟嘌呤（OG）的结果，本项目研究的过度氧化鸟嘌呤病变引起的进一步氧化的OG。这些病变构成了一个高度致突变的乙内酰脲结构家族，包括螺亚氨基二乙内酰脲（Sp）和胍基乙内酰脲（Gh），它们似乎除了引起G至T突变外，还引起G至C突变。在本工作中，这类化合物的相关成员将进行研究，以进一步探索氧化途径，产物结构和活性与DNA加工酶，包括碱基切除修复和核苷酸切除修复机制之间的关系。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，可能会解释额外的观察氧化应激的诱变谱，包括双链串联病变的形成。这项工作的具体目标是：（1）通过使用合成的含有损伤的寡脱氧核苷酸、质谱和X射线晶体学，解决围绕双链体DNA中乙内酰脲损伤形成的结构问题，（二）通过体外和体内生物化学测定，了解碱基切除修复与核苷酸切除修复对乙内酰脲损伤的作用，特别关注Nei-如（hNEIL 1）和Fpg糖基化酶，和（3）开发创新的测定，通过产生与荧光读数偶联的DNA损伤的适体来检测细胞裂解物中的乙内酰脲产物。 公共卫生相关性：氧化应激在DNA损伤中起着重要作用，DNA损伤是癌症、衰老、神经和心脏疾病的基础。该项目将有助于确定特定的氧化DNA碱基和碱基加合物如何导致与癌症相关的突变。此外，还将开发监测这些受损碱基从DNA中形成和切除的方法，揭示有关DNA损伤与癌症之间关系的新信息。

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.