Mechanisms of the 3'-->5' deoxyribonucleases

描述(申请人提供)：3‘？5’脱氧核糖核酸酶是DNA新陈代谢中必不可少的酶，它催化从DNA的3‘末端切除核苷酸，为DNA加工的后续步骤准备这些3’末端。该项目的长期目标是了解由TREX基因编码的在人类细胞中起作用的3‘？5’脱氧核糖核酸酶的生物化学，并阐明这些酶发挥作用的DNA代谢途径。人类细胞中存在3‘脱氧核糖核酸酶已有近40年的历史，但这些酶的生物学功能尚未得到很好的描述。在之前的资助期间，我们确定了TREX1和TREX2酶的结构，使我们能够开始描述这些3‘脱氧核糖核酸酶的生化性质。TREX1和TREX2基因编码两个结构相似的二聚体3‘脱氧核糖核酸酶。TREX1酶含有一个在TREX2中没有的C-末端区域，并且只在哺乳动物中发现两个TREX基因。我们对TREX1精确分子特征的生化研究使我们能够量化在自身免疫性疾病Aicardi-Goutieres综合征、家族性冻疮狼疮和一些系统性红斑狼疮患者中发现的突变TREX1酶的活性。在自身免疫性疾病患者中发现TREX1突变，这与TREX1参与细胞凋亡死亡的发现是平行的。在这次竞争性更新中，我们将利用我们在前一个资助期开发的分子工具，重点研究TREX1在细胞死亡途径中的作用机制。这些研究将通过确定TREX1的生理底物进一步加深我们对TREX1核酸外切酶在细胞内DNA处理的理解，并将向我们提供TREX1功能障碍导致与自身免疫性疾病相关的病理结果的机制。我们努力了解TREX1的生物化学和细胞功能的机制，根据以下目标。在目标1中，我们将确定TREX1在催化核心内的作用机制。在目标2中，我们将确定TREX1在细胞死亡途径中对染色质分解的影响。在目标3中，我们将确定TREX1靶向内质网的机制。 公共卫生评论：这项拨款提案是为了研究酶是如何处理DNA的，并确定这些酶的功能障碍如何导致自身免疫性疾病。选举的结果是 提出的研究将对系统性红斑狼疮等复杂自身免疫性疾病的医疗产生重大影响。

DESCRIPTION (provided by applicant): The 3'??5' deoxyribonucleases are essential enzymes in DNA metabolism that catalyze excision of nucleotides from the 3' ends of DNA to prepare these 3' termini for subsequent steps during DNA processing. The long term goal of this project is to understand the biochemistry of the 3'??5' deoxyribonucleases encoded by the TREX genes that function in human cells and to elucidate the DNA metabolic pathways in which these enzymes function. The existence of 3' deoxyribonucleases in human cells has been recognized for almost forty years, but the biological functions of these enzymes are not well described. In the previous funding period we determined the structures of the TREX1 and TREX2 enzymes allowing us to begin to describe the biochemical properties of these 3' deoxyribonucleases. The TREX1 and TREX2 genes encode two structurally similar dimeric 3' deoxyribonucleases. The TREX1 enzyme contains a C-terminal region that is not found in TREX2, and the presence of two TREX genes is only found in mammals. Our biochemical studies to define the precise molecular characteristics of TREX1 have allowed us to quantify the activities of mutant TREX1 enzymes identified in patients diagnosed with the autoimmune diseases Aicardi- Goutieres syndrome, familial chilblain lupus, and in some cases of systemic lupus erythematosus. The finding of TREX1 mutations in autoimmune disease patients parallels the finding of TREX1 participation in apoptotic cell death. In this competitive renewal we will utilize the molecular tools we have developed in the previous funding period to focus our studies on the mechanisms of TREX1 action within cell death pathways. These studies will further our understanding of TREX1 exonuclease processing of DNA in cells by identifying the physiological substrate of TREX1 and will inform us of the mechanisms of TREX1 dysfunction that cause the pathological findings associated with autoimmune disease. Our efforts to understand the mechanisms of TREX1 biochemistry and cellular function are addressed according to the following aims. In Aim 1 we will determine the mechanism of TREX1 action within the catalytic core. In Aim 2, we will determine the effects of TREX1 on chromatin disassembly in cell death pathways. In Aim 3 we will determine the mechanism of TREX1 targeting to the ER. PUBLIC HEALTH REVELANCE: This grant proposal is to study how enzymes process DNA and to determine how dysfunction of these enzymes causes autoimmune disease. The outcome of the proposed research will have a significant impact on the medical treatment of complex autoimmune diseases such as systemic lupus erythematosus.