Discovering TREX1 and TREX2 Function in Mammalian Cells and Mice

描述(由申请人提供)：我们的假设是TREX1和TREX2是具有3‘-5’切割活性的外切酶，这对于在DNA复制和/或DNA修复过程中去除有问题的核苷酸非常重要，我们的目标是在细胞和小鼠中研究这些蛋白质。目前，TREX1和TREX2被认为是外切酶，基于它们与大肠杆菌DNA pol1大片段和噬菌体T4 DNA pol三个核酸外切酶序列基序的同源性以及它们在体外的3‘-5’外切酶活性。然而，目前还没有更多的公开信息来定义它们的细胞功能。因此，TREX1和TREX2对于维持基因组稳定和预防癌症具有潜在的重要意义。有三个目标定义了TREX1和TREX2。目的1：阐明TREX1和TREX2的基本生化功能。已知有三种生化功能：1)自结合；2)核酸外切酶活性；3)DNA结合活性。我们已经建立了体外实验来观察这些活动，我们的目标是产生受损形式的TREX1和TREX2，它们执行一些但不是所有这些活动。目的：通过对转基因小鼠胚胎干细胞的分析，发现TREX1和TREX2的生物学意义。我们将比较一个空突变和在目标1中发现的改变某些但不是所有功能的突变。此时，我们已经产生了TREX2缺失的ES细胞，我们的初步结果表明TREX2确实参与了基因组的维持。我们发现，TREX2缺失的细胞对一些DNA损伤剂的敏感性发生了变化，并且TREX2缺失的细胞表现出基因组的不稳定性，包括总的染色体重排。目的：分析野生型和P53突变背景下的TREX基因缺失小鼠。我们将进行寿命分析，并确定年龄相关特征的发病、发病率和谱系，包括癌症和基因组不稳定。通过对双突变小鼠的研究，将确定P53介导的对受损DNA的反应的影响。这一建议的完成将极大地促进我们对TREX1和TREX2在维持基因组稳定和预防癌症方面的理解。

DESCRIPTION (provided by applicant): Our hypothesis is that TREX1 and TREX2 are exonucleases with 3'-"5' excision activity that is important for removing problematic nucleotides during either DNA replication and/or DMA repair and our goal is to study these proteins in cells and mice. Currently TREX1 and TREX2 are thought to be exonucleases based on their homology to three exonuclease sequence motifs of E. coli DNA pol 1 large fragment and bacteriophage T4 DNA pol and based on their 3'-"5' exonuclease activity in vitro. However, at this time there is no more published information defining their cellular function. Thus, TREX1 and TREX2 are potentially important for maintaining genomic stability and perhaps cancer prevention. There are three aims that define TREX1 and TREX2. Aim 1: to elucidate fundamental TREX1 and TREX2 biochemical functions. There three known biochemical functions: 1) self association 2) exonuclease activity, 3) and DNA binding activity. We have setup in vitro assays to observe these activities and our goal is to generate impaired forms of TREX1 and TREX2 that perform some but not all of these activities. Aim 2: to discover the biological importance of TREX1 and TREX2 by analyzing genetically altered mouse embryonic stem (ES) cells. We will compare a mutation that is null to mutations that alter some but not all functions as discovered in aim 1. At this time we have generated TREX2-null ES cells and our preliminary results demonstrate that TREX2 is indeed involved in genome maintenance. We show TREX2-null cells exhibit altered sensitivity to some DNA damaging agents and TREX2-null cells exhibit genomic instability including gross chromosomal rearrangements. Aim 3: to analyze TREX-null mice in wild type and p53 mutant backgrounds. We will perform a life span analysis and determine the onset, incidence and spectra of age-related characteristics including cancer and genomic instability. The impact p53-mediated responses to damaged DNA will be determined by studying double-mutant mice. Completion of this proposal will greatly facilitate our understanding of TREX1 and TREX2 for maintaining genome stability and for preventing cancer.