Chemistry and Biology of DNA Ligation

当前奖项摘要 这项提案的总体目标是确定人类DNA的机制和特异性 连接酶。所有生物体都有DNA复制和DNA修复的绝对需求， 以合成新的细胞并维持正确的细胞功能。鉴于其中央 重要的是，人们对研究这些途径很感兴趣。DNA连接酶是 对于DNA复制和大多数DNA修复途径至关重要，然而， 关于DNA连接酶如何起作用的基本问题，我们现有的模型远远落后于 那些DNA聚合酶可用的。我们认为，学习 人类DNA连接酶的分子机制以及从这一奋进中获得的见解 将对人类健康有重大价值。这些知识可能存在于 在患有DNA连接酶功能缺陷和处于异常状态的患者中是有用的， 连接酶已经过表达，例如癌症。我们将使用定量机制 酶学技术来表征人DNA连接酶1（LIG1）和DNA连接酶3（LIG3）。 遗传和细胞观察表明，这些酶共享一些冗余的生物学特性， 功能，但每个基因产物存在不同的功能。我们将延续我们以前的 对LIG1的动力学和结构研究，以了解这种酶的特异性，并确定 定位和接合单股断裂的步骤最少。这项工作将得到以下方面的支持： 另外的晶体结构将表征酶结合的中间体， 以前的特点。我们最近成功地生产了大量的 重组LIG 3 α和β亚型，我们将进行动力学和热力学分析。 表征，以了解与LIG1的相似性和差异。对于这两种酶，我们将 使用定点诱变来靶向特定功能，如金属结合，DNA结合， 和催化活性。对这些突变蛋白的分析将提供对这些突变蛋白的理解。 真核生物DNA连接的分子特征，这将是非常宝贵的了解连接酶 在正常细胞和人类疾病中的功能

Abstract of Current Award The overall 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. Given its central importance, there is a great deal of interest in studying these pathways. DNA ligases are essential for DNA replication and most DNA repair pathways, however there are many fundamental questions about how DNA ligases function and our existing models lag far behind those that are available for DNA polymerases. We believe that it is essential to learn the molecular mechanism of human DNA ligases and that the insights gained from this endeavor will have significant value to human health. Some examples of where this knowledge could be useful is in patients that suffer deficiency in DNA ligase function and in abnormal states in which ligases have been overexpressed such as cancer. We will use quantitative mechanistic enzymology techniques to characterize human DNA ligase 1 (LIG1) and DNA ligase 3 (LIG3). Genetic and cellular observations suggest that these enzymes share some redundant biological functions, but that distinct functions exist for each gene product. 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. This work will be supported by additional crystal structures that will characterize the enzyme-bound intermediates that have not been previously characterized. We have recently succeeded in producing large quantities of recombinant LIG 3 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 binding, DNA binding, and catalytic activity. 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