Chemistry and Biology of DNA Ligation

这项建议的目的是确定人类DNA连接酶的机制和特异性。所有生物体 对DNA复制和DNA修复有绝对要求，以合成新细胞和 保持正确的细胞功能。DNA连接酶催化DNA复制的终极步骤，大多数DNA 修复途径，然而，大多数研究都集中在DNA聚合酶上，因此我们缺乏 对DNA连接酶的机制和特性有基本的了解。学习分子是很重要的。 人类DNA连接酶的机制，这些见解将对理解人类 健康。我们的工作是以已知的DNA修复和复制生物学为指导的，我们专注于未回答 无法用目前对这些途径的理解来解释的谜题。几个近期的临床案例 这种知识的关键是在DNA连接酶基因遗传突变的患者中，例如LIG1 导致原发免疫缺陷的综合征和导致严重合并的LIG4综合征 免疫缺陷(SCID)和异常状态，如癌症，其中连接酶，特别是LIG3， 被过度表达了。我们将使用定量机械酶学和x射线结晶学(in 与Scott Williams博士合作)以确定人类DNA连接酶1(LIG1)和DNA连接酶3(LIG3)的特征。 遗传观察表明，这些酶具有独特的功能，但有一些相似之处。为 例如，LIG1和LIG3都在细胞核内的DNA修复和复制中发挥作用，但只有LIG3在 线粒体。我们将扩展我们之前对LIG1的动力学和结构的研究，以了解LIG1的特异性 确定定位和接合单链断裂的最小步骤。我们最近做了 成功地生产了大量的重组LIG3α和β亚型，我们将进行 动力学和热力学表征，以了解与LIG1的异同。对两个人都是 酶，我们将使用定点突变来靶向特定的功能，例如金属辅因子结合， DNA结合和催化专一性。对这些突变蛋白的分析将提供对 真核DNA连接的分子特征对了解正常人连接酶功能具有重要意义 细胞和人类疾病中。该基金的核心目标是从分子水平上理解 LIG1和LIG3的机制和特异性，但随后出现的机制模型将在 适当的单元格。这项工作将揭示不同DNA修复途径之间的相互联系 为了解监管相互作用和修改提供了坚实的基础。

The goal of this proposal is to determine the mechanism and specificity of human DNA ligases. All organisms have an absolute requirement for DNA replication and DNA repair in order to synthesize new cells and to maintain correct cellular functions. DNA ligases catalyze the ultimate step in DNA replication and most DNA repair pathways, however most research has focused on DNA polymerases and therefore we lack a fundamental understanding of DNA ligase mechanisms and specificities. It is essential to learn the molecular mechanism of human DNA ligases and that these insights will have significant value for understanding human health. Our work is guided by the known biology of DNA repair and replication and we focus on unanswered puzzles that cannot be explained by current understanding of these pathways. Some recent clinical examples of where this knowledge is critical is in patients with inherited mutations in DNA ligase genes, such as LIG1 syndrome which causes a Primary ImmunoDeficiency and LIG4 syndrome which causes Severe Combined ImmunoDeficiency (SCID), and in abnormal states such as cancer in which ligases, especially LIG3, have been overexpressed. We will use quantitative mechanistic enzymology and x-ray crystallography (in collaboration with Dr. Scott Williams) to characterize human DNA ligase 1 (LIG1) and DNA ligase 3 (LIG3). Genetic observations suggest that these enzymes have unique functions, but share some similarities. For example, LIG1 and LIG3 both function in DNA repair and replication in the nucleus, but only LIG3 functions in the mitochondria. We will extend our previous kinetic and structural studies of LIG1 to understand specificity of this enzyme and to define the minimal steps in locating and engaging a single strand break. We have recently succeeded in producing large quantities of recombinant LIG3 alpha and beta isoforms and we will perform a kinetic and thermodynamic characterization to understand similarities and differences with LIG1. For both enzymes, we will use site-directed mutagenesis to target specific functions, such as metal cofactor binding, DNA binding, and catalytic specificity. Analysis of these mutant proteins will provide an understanding of the molecular features of eukaryotic DNA ligation that will be invaluable to understand ligase function in normal cells and in human disease. The core objectives of the grant are to develop a molecular understanding of the mechanism and specificity of LIG1 and LIG3, but the mechanistic models that emerge will then be tested in an appropriate cell. This work will uncover the interconnections between different DNA repair pathways and provide a strong foundation for understanding regulatory interactions and modifications.