Chemistry and Biology of DNA Ligation

该提案的目标是确定人类DNA连接酶的机制和特异性。所有生物体 为了合成新的细胞， 保持正常的细胞功能。DNA连接酶催化DNA复制的最终步骤， 修复途径，然而，大多数研究都集中在DNA聚合酶，因此我们缺乏 对DNA连接酶的机制和特异性有基本的了解。学习分子生物学 人类DNA连接酶的机制，这些见解将有重大价值，了解人类 健康我们的工作是由DNA修复和复制的已知生物学指导的，我们专注于未回答的问题。 目前对这些途径的理解无法解释的谜题。最近的一些临床例子 这些知识在DNA连接酶基因遗传突变的患者中至关重要，如LIG1 引起原发性免疫缺陷综合征和引起严重联合免疫缺陷综合征的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。我们将使用定量机械酶学和X射线晶体学（在 与Scott威廉姆斯博士合作）来表征人DNA连接酶1（LIG1）和DNA连接酶3（LIG3）。 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都在细胞核中的DNA修复和复制中起作用，但只有LIG3在细胞核中起作用。 线粒体我们将扩展我们以前对LIG1的动力学和结构研究，以了解LIG1的特异性。 这种酶，并确定在定位和从事单链断裂的最小步骤。我们最近 成功地生产了大量的重组LIG3 α和β亚型，我们将进行一项 动力学和热力学表征，以了解与LIG1的相似性和差异。为 酶，我们将使用定点诱变靶向特定功能，如金属辅因子结合， DNA结合和催化特异性。对这些突变蛋白的分析将提供对这些突变蛋白的理解。 真核DNA连接的分子特征对于了解正常细胞中连接酶的功能非常宝贵 细胞和人类疾病。该基金的核心目标是从分子水平上了解 LIG1和LIG3的机制和特异性，但出现的机制模型将在一个 合适的细胞。这项工作将揭示不同DNA修复途径之间的相互联系， 为理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.