DNA Repair and Replication: Fundamental Studies and Applications

描述（由申请方提供）：DNA持续暴露于内源性和外源性试剂，这些试剂通过修饰其核碱基产生损伤。体内DNA中这些病变的存在与衰老、疾病如癌症和其他遗传疾病有关。但是，DNA损伤也可能是有益的。例如，电离辐射是用于治疗癌症的最常见的非手术方法。辐射通过破坏DNA杀死肿瘤细胞。这项研究的目的是了解氧化应激导致的DNA损伤是如何修复、复制和反应形成其他损伤的。 我们的努力集中在氧化脱碱基病变，这是不能形成沃森-克里克氢键。与先前所认为的相反，氧化的脱碱基损伤以彼此不同的方式与聚合酶相互作用，并且与核苷酸糖苷键的正式水解产生的脱碱基位点（AP）相互作用。因此，不能形成沃森-克里克氢键并不意味着病变是无意义的。我们将使用合成化学来合成病变，快速淬灭动力学来确定聚合酶机制，机械研究来确定修复的不可逆抑制，大分子NMR来确定结构，并在酵母中进行诱变实验来确定单个病变的特性。 此外，我们将研究氧化脱碱基病变如何在核小体中反应。核小体是否影响病变的反应性？它们会形成DNA-蛋白质交联吗？对核小体的研究使我们离研究细胞中的DNA损伤又近了一步。我们也将设计研究方法，利用光酸结合激光光解产生损伤来研究细胞中脱碱基位点的DNA修复。 我们还在开发修饰的核苷酸，它们将成为DNA聚合酶B的不可逆抑制剂，以及在肿瘤细胞中过度表达的碱基切除修复中的重要酶。 总之，该项目结合了有机化学，生物化学和生物学，旨在改善生物体中发生的重要化学过程。

DESCRIPTION (provided by applicant): DNA is constantly exposed to endogenous and exogenous agents that produce lesions by modifying its nucleobases. The presence of these lesions in DNA in vivo is associated with aging, diseases such as cancer, and other genetically based diseases. However, DNA damage can also be beneficial. For instance, ionizing radiation is the most common nonsurgical method used to treat cancer. Radiation kills tumor cells by damaging DNA. The goals of this research are to understand how lesions produced in DNA as a result of oxidative stress are repaired, replicated, and react to form other lesions. Our effort is focused on oxidized abasic lesions, which are incapable of forming Watson- Crick hydrogen bonds. Contrary to what was previously believed, oxidized abasic lesions interact with polymerases in distinct ways from each other and from an abasic site (AP) resulting from formal hydrolysis of a nucleotide's glycosidic bond. Hence, the inability to form Watson- Crick hydrogen bonds does not mean that a lesion is noninstructive. We will use synthetic chemistry to synthesize lesions, rapid-quench kinetics to determine polymerase mechanisms, mechanistic studies to determine irreversible inhibition of repair, macromolecular NMR to determine structure, and carry out mutagenesis experiments in yeast to determine the properties of the individual lesions. In addition, we will investigate how oxidized abasic lesions react in nucleosomes. Do nucleosomes affect the reactivity of the lesions? Do they form DNA-protein cross-links? Studies in nucleosomes bring us one step closer to studying DNA damage in cells. We will also design methods for studying DNA repair of abasic sites in cells by using photoacids in conjunction with laser photolysis to produce the lesions. We are also developing modified nucleotides that will be irreversible inhibitors of DNA polymerase b, and important enzyme in base excision repair that is over expressed in tumor cells. In summary, the project combines organic chemistry, biochemistry, and biology with the goal of improving fundamentally important chemical processes that occur in living organisms.