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 DMArepair 过程中去除有问题的核苷酸很重要 我们的目标是研究细胞和小鼠中的这些蛋白质。目前TREX1和TREX2被认为是 基于与大肠杆菌 DNA pol 1 的三个核酸外切酶序列基序的同源性的核酸外切酶 大片段和噬菌体 T4 DNA pol 并基于其体外 3'-¿5' 核酸外切酶活性。 然而，目前还没有更多公开的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性甚至癌症可能很重要 预防。定义 TREX1 和 TREX2 的三个目标。目标1：阐明基本原理 TREX1 和 TREX2 生化功能。已知三种生化功能：1) 自身 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测定 观察这些活动，我们的目标是生成受损形式的 TREX1 和 TREX2 执行部分但不是全部这些活动。目标 2：发现 TREX1 的生物学重要性 TREX2 通过分析基因改变的小鼠胚胎干 (ES) 细胞。我们将比较一个 对于改变目标 1 中发现的部分但不是全部功能的突变来说，该突变无效。 当我们生成 TREX2-null ES 细胞时，我们的初步结果表明 TREX2 是 确实参与了基因组维护。我们发现 TREX2 缺失细胞表现出对 一些 DNA 损伤剂和 TREX2 缺失细胞表现出基因组不稳定性，包括总体不稳定性 染色体重排。目标 3：分析野生型和 p53 突变型 TREX 缺失小鼠 背景。我们将进行寿命分析并确定疾病的发作、发生率和谱 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。完成此 该提案将极大地促进我们对 TREX1 和 TREX2 维持基因组的理解 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.