DNA Adduct-Induced Mutagenesis

这项建议集中在三种类型的化学和结构生物学 DNA损伤。丙二醛(MDA)，由脂质过氧化和 前列腺素的生物合成，与DNA反应形成一种被称为OPdG的加合物，它与人类癌症有关。黄曲霉毒素B(AFB1)是一种高度致突变的真菌毒素，是一种食品污染物。流行病学证据表明，它与乙肝病毒协同作用，促进肝癌；这些癌症通常表现出p53肿瘤抑制基因的突变。AFB1的细胞色素P450氧化产物使脱氧鸟苷的N7原子烷化。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(胸腺嘧啶二醇；Tg)。TG通过原核和真核DNA聚合酶抑制复制，并与人类癌症的病因有关，可能与DNA双链断裂的产生有关。强调这三种类型的DNA损伤是基于这样的观察，即当存在于DNA中时，每一种类型的损伤都可以进行进一步的化学处理，这取决于DNA是以双链还是单链的形式存在，也取决于双链DNA中互补核苷酸的序列和同一性。因此，在每种情况下，对损伤的生物反应预计都取决于其在DNA中的下游化学。一些继发性病变，例如AFB1-N7-DG Fapy病变，比最初的病变更有害。本研究旨在描绘DNA中这些损伤的复杂化学，并定义潜在的结构-活性关系，以努力了解它们与DNA处理酶的相互作用，例如损伤旁路聚合酶。为了实现这一点，将结合使用生化和生物物理方法。核磁共振将被用来确定寡聚脱氧核苷酸中定点DNA损伤的结构。结晶学将被用来确定DNA聚合酶复合体中特定部位受损的寡核苷酸的结构。由此产生的结构性数据将与范德比尔特大学和康涅狄格大学的同事合作进行解释。最终目标是了解复杂的细胞对特定类型的DNA损伤的反应。

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.