Discovering TREX1 and TREX2 Function in Mammalian Cells and Mice

我们的假设是TREX1和TREX2是具有3‘-5’切除活性的核酸外切酶，即 对于在DNA复制和/或DNA修复期间移除有问题的核苷酸以及 我们的目标是在细胞和小鼠中研究这些蛋白质。目前认为TREX1和TREX2是 核酸外切酶与大肠杆菌DNAPol1三个核酸外切酶序列基序的同源性 大片段和T4噬菌体DNA Poll，并基于它们的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癌症具有潜在的重要意义 预防。有三个目标定义了TREX1和TREX2。目标1：阐明基本原理 TREX1和TREX2具有生化功能。已知有三种生化功能：1)自身 关联2)核酸外切酶活性，3)DNA结合活性。我们已经建立了体外检测来 观察这些活动，我们的目标是产生受损形式的TREX1和TREX2 执行这些活动中的一些，但不是全部。目的2：发现TREX1的生物学重要性 和TREX2通过分析转基因小鼠胚胎干细胞。我们将比较一个 与目标1中发现的改变某些但不是全部功能的突变相比为空的突变。 我们已经生成了TREX2空的ES细胞，并且我们的初步结果表明TREX2是 确实参与了基因组的维护。我们显示TREX2-空细胞表现出对 一些DNA损伤剂和TREX2缺失细胞表现出基因组不稳定，包括 染色体重排。目的3：分析野生型和突变型TREX基因缺失小鼠 背景。我们将进行寿命分析，并确定发病、发病和光谱 与年龄相关的特征，包括癌症和基因组不稳定。P53介导的影响 对受损DNA的反应将通过研究双突变小鼠来确定。完成这项工作 该建议将极大地促进我们对TREX1和TREX2保持基因组的理解 稳定和预防癌症。

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 DMArepair 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. Wewill 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 preventingcancer.