DNA Repair and Replication: Fundamental Studies and Applications

描述（由申请人提供）：DNA不断暴露于内源性和外源性物质，通过修饰其核碱基产生病变。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用的非手术方法。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在DNA中产生的损伤是如何修复、复制和反应形成其他损伤的。我们的努力集中在氧化基损伤，这是不能形成沃森-克里克氢键。与之前所认为的相反，氧化基灶以不同的方式与聚合酶相互作用，并与碱基位点（AP）相互作用，这是由核苷酸的糖苷键的形式水解产生的。因此，不能形成沃森-克里克氢键并不意味着病变没有指导意义。我们将使用合成化学来合成病变，快速淬火动力学来确定聚合酶的机制，机理研究来确定不可逆的修复抑制，大分子核磁共振来确定结构，并在酵母中进行诱变实验来确定单个病变的性质。此外，我们将研究氧化基性病变在核小体中的反应。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？核小体的研究使我们离研究细胞中的DNA损伤又近了一步。我们还将设计方法，通过使用光酸结合激光光解来产生病变，研究细胞中基本位点的DNA修复。我们还在开发修饰的核苷酸，它将成为DNA聚合酶b的不可逆抑制剂，DNA聚合酶b是肿瘤细胞中过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，目的是改善生物体内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. PUBLIC HEALTH RELEVANCE: DNA damage and repair are important chemical processes that significantly impact human health. These chemical processes are associated with aging and a variety of diseases, such as cancer. Understanding the chemistry, biochemistry, and biological effects of damaged DNA enhances our molecular level understanding of the etiology of diseases, such as cancer, as well as the various treatments for which nucleic acids are the target.