DNA Repair and Replication: Fundamental Studies and Applications

描述(申请人提供)：DNA经常暴露在内源性和外源性因素中，这些因素通过修改其核苷酸碱基来产生损伤。体内DNA中这些损伤的存在与衰老、癌症等疾病和其他基于基因的疾病有关。然而，DNA损伤也可能是有益的。例如，电离辐射是治疗癌症最常见的非手术方法。辐射通过破坏DNA来杀死肿瘤细胞。这项研究的目标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。我们的工作重点是氧化的基本损伤，这些损伤不能形成Watson-Crick氢键。与以前认为的相反，氧化的碱性损伤以不同的方式与聚合酶相互作用，并且来自核苷酸的糖苷键的形式水解产生的碱性位点(AP)。因此，不能形成Watson-Crick氢键并不意味着病变没有指导性。我们将使用合成化学来合成损伤，快速猝灭动力学来确定聚合酶机制，机制研究来确定修复的不可逆抑制，大分子核磁共振来确定结构，并在酵母中进行突变实验来确定单个损伤的性质。此外，我们还将研究核小体中氧化的基本病变是如何反应的。核小体是否影响病变的反应性？它们会形成DNA-蛋白质交联链吗？对核小体的研究使我们离研究细胞中的DNA损伤更近了一步。我们还将设计方法来研究细胞中基本位置的DNA修复，方法是结合使用光酸和激光光解来产生损伤。我们还在开发修饰的核苷酸，它将是DNA聚合酶b的不可逆抑制物，以及在肿瘤细胞中过度表达的碱基切除修复中的重要酶。总而言之，该项目结合了有机化学、生物化学和生物学，目标是从根本上改善发生在生物体内的重要化学过程。

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.