Discovering TREX1 and TREX2 Function in Mammalian Cells and Mice

我们的假设是TREX 1和TREX 2是具有3 '-<$5'切除活性的核酸外切酶， 对于在DNA复制和/或DMA修复期间去除有问题的核苷酸是重要的， 我们的目标是在细胞和小鼠中研究这些蛋白质。目前，TREX 1和TREX 2被认为是 基于它们与E.大肠杆菌DNA pol 1 大片段和噬菌体T4 DNA pol并基于它们体外3 ′-5 ′核酸外切酶活性。 然而，目前还没有更多的公开信息定义它们的细胞功能。因此，在本发明中， TREX 1和TREX 2对维持基因组稳定性和可能的癌症具有潜在的重要性 预防有三个目标定义了TREX 1和TREX 2。目标1：阐明基本原理 TREX 1和TREX 2的生物化学功能。已知有三种生物化学功能：1）自我 关联2）核酸外切酶活性，3）和DNA结合活性。我们已经建立了体外试验， 观察这些活动，我们的目标是产生TREX 1和TREX 2的受损形式， 执行部分但不是全部这些活动。目的2：发现TREX 1的生物学重要性 和TREX 2通过分析遗传改变的小鼠胚胎干细胞（ES细胞）。我们将比较一个 从无效突变到目标1中发现的改变某些但不是全部功能的突变。在这个 我们已经产生了TREX 2-null ES细胞，我们的初步结果表明TREX 2是 确实参与了基因组的维护。我们发现，TREX 2-null细胞表现出对 一些DNA损伤剂和TREX 2缺失细胞表现出基因组不稳定性， 染色体重排目的3：分析野生型和p53突变型TREX基因敲除小鼠 背景我们将进行寿命分析，并确定发病，发病率和光谱 与年龄相关的特征，包括癌症和基因组不稳定性。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。完成本 这一建议将大大有助于我们理解TREX 1和TREX 2在维持基因组中的作用 稳定性和预防癌症。

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.