DNA Adduct-Induced Mutagenesis

该提案侧重于三种类型的化学和结构生物学， DNA损伤。脂质过氧化反应产生的丙二醛（MDA）， 前列腺素的生物合成，与DNA反应形成被称为OPdG的加合物，其与人类癌症有关。黄曲霉毒素B（AFB 1）是一种高度致突变的真菌毒素，是食品污染物。流行病学证据表明，它与B型肝炎病毒协同作用，促进肝癌;这些癌症往往表现出p53肿瘤抑制基因的突变。黄曲霉毒素B1的细胞色素P450氧化产物烷基化脱氧鸟苷的N7原子。电离辐射对胸腺嘧啶的氧化损伤产生5，6-二羟基-二氢-2 '-胸腺嘧啶（胸腺嘧啶乙二醇; Tg）。Tg抑制原核和真核DNA聚合酶的复制，并且与人类癌症的病因学有关，可能与DNA中双链断裂的产生有关。对这三种类型的DNA损伤的强调是基于这样的观察，即当存在于DNA中时，这些类型的损伤中的每一种都可以经历进一步的化学作用，这取决于DNA是以双链体形式还是以单链形式存在，并且还取决于双链体DNA中互补核苷酸的序列和身份。因此，在每种情况下，对病变的生物学反应预计取决于其下游DNA化学。一些继发性病变;例如，AFB 1-N7-dG FAPy损伤比初始损伤更有害。这项研究旨在描述这些DNA损伤的复杂化学性质，并确定潜在的结构-活性关系，以了解它们与DNA加工酶（如损伤旁路聚合酶）的相互作用。为了实现这一目标，将利用生物化学和生物物理方法的组合。核磁共振将用于确定寡脱氧核苷酸中位点特异性DNA损伤的结构。晶体学将用于确定与DNA聚合酶复合的位点特异性损伤的寡脱氧核苷酸的结构。由此产生的结构数据将与范德比尔特大学和康涅狄格大学的同事合作解释。最终目标是了解细胞对特定类型DNA损伤的复杂反应。

This proposal focuses upon the chemical and structural biology of three types of DNA damage. Malondialdehyde (MDA), produced by lipid peroxidation and prostaglandin biosynthesis, reacts with DNA to form an adduct known as OPdG, which is implicated in human cancer. Aflatoxin B (AFB1), a highly mutagenic mycotoxin, is a contaminant of food. Epidemiological evidence suggests that it acts synergistically with the hepatitis B virus to promote hepatic cancer; these cancers often exhibit mutations in the p53 tumor suppressor gene. The cytochrome P450 oxidation product of AFB1 alkylates the N7 atom of deoxyguanosine. Oxidative damage to thymine from ionizing radiation produces 5,6-dihydroxy-dihydro-2'-thymine (thymine glycol; Tg). Tg inhibits replication by prokaryotic and eukaryotic DNA polymerases, and is implicated in the etiology of human cancer, perhaps associated with the generation of double strand breaks in DNA. The emphasis on these three types of DNA damage is predicated upon the observation that when present in DNA, each of these types of damage can undergo further chemistry depending upon whether the DNA exists in duplex or in single-stranded form, and also depending upon the sequence and identity of the complementary nucleotide in duplex DNA. Thus, in each case the biological response to the lesion is anticipated to depend upon its downstream chemistry in DNA. Some of the secondary lesions; e.g., the AFB1-N7-dG FAPy lesions, are more deleterious than are the initial lesions. This research seeks to delineate the complex chemistry of these lesions in DNA, and to define underlying structure-activity relationships, in an effort to understand their interactions with DNA processing enzymes, such as damage bypass polymerases. To accomplish this, a combination of biochemical and biophysical approaches will be utilized. NMR will be used to determine the structures of site-specific DNA damage in oligodeoxynucleotides. Crystallography will be used to determine structures of sitespecifically damaged oligodeoxynucleotides in complex with DNA polymerases. The resulting structural data will be interpreted in collaboration with colleagues at Vanderbilt University and the University of Connecticut. The ultimate goal is to understand complex cellular responses to specific types of DNA damage.