Genotoxic stress response and mutagenesis in normal tissues of mice deficient in homology directed repair

项目总结/摘要 该项目的长期目标是描述维持生存能力的途径和机制 以及暴露于环境遗传毒性压力的成年哺乳动物的基因组完整性。DNA损伤可能是 通过高保真途径同源定向修复（HDR）或通过易错非同源末端修复 加入（NHEJ）。成年脊椎动物中HDR缺失的后果尚不清楚， 基因敲除是胚胎致死的，因此，我们的大多数理解来自细胞培养模型或推断 由HDR缺陷引起的肿瘤。 为了解决这一关键需求，我们已经产生了基因工程小鼠， 在发育后的每个组织中，高达99%的必需HDR基因Brca 1。我们将利用这一模式， 解决了以下关键问题：1）HDR在不同类型的DNA后细胞/组织/生物体活力中的作用 损伤; 2）不同组织对HDR的依赖性，以在不同类型的DNA损伤后维持基因组完整性。 损伤; 3）由不同应激引起的HDR缺陷小鼠的组织中的突变特征，允许推断 4）转录因子p53如何与HDR在每个细胞中协同作用; 5）HDR和NHEJ在DNA修复中的协同作用。我们假设， 由于缺失了必需的HDR基因Brca 1，已知发展为HDR缺陷型肿瘤的组织将具有较高的 突变率和p53介导的阻滞程序优于细胞凋亡。 具体目标1。确定HDR是如何通过介导组织特异性细胞和 转录反应。在野生型或Brca 1缺失的小鼠中，我们将研究Brca 1/HDR如何保留 不同类型DNA损伤后的近期存活率通过3种措施：1）DNA损伤反应和细胞 命运（凋亡、细胞周期停滞）; 2）转录组学变化; 3）不同组织的病理学。 具体目标2。确定Brca 1/HDR在维持基因组中的组织和DNA损伤特异性作用 完整在这个目标中，我们将研究HDR缺陷对基因组维持的长期后果。 已知体细胞突变随着年龄的增长在正常人体组织中积累，并与疾病有关。我们 将研究修复的保真度和小鼠的寿命，有或没有功能性HDR，生存不同类型的 DNA损伤。深度测序和突变特征分析将揭示哪些组织依赖HDR， 在不同的HDR缺陷组织中，一种类型的DNA扰动是最具致突变性的。 具体目标3。测定NHEJ缺陷或NHEJ和HDR缺陷小鼠的生存力和应激反应。 由于胚胎中Brca 1或NHEJ基因Lig 4（编码连接酶4）的缺失是致命的，因此他们的个体 在成体组织中的协同作用尚不清楚。在这个目标中，我们将研究生存和应激反应 在成年小鼠中缺失HDR和NHEJ途径后，仅留下SSA和alt-NHEJ。

Project Summary/Abstract The long-term objective of this project is to characterize the pathways and mechanisms that preserve viability and genome integrity in adult mammals exposed to environmental genotoxic stresses. DNA damage can be repaired by the high fidelity pathway homology directed repair (HDR) or by error prone non-homologous end joining (NHEJ). The consequences of HDR loss in adult vertebrates is not well understood because germline knockouts are embryonic lethal, thus, most of our understanding has come from cell culture models or inferred from tumors caused by HDR deficiency. To address this critical need, we have generated genetically engineered mice that can undergo inducible deletion of up to 99% of the essential HDR gene Brca1 in every tissue, post development. We will use this model to address critical questions on 1) the role of HDR in cell/tissue/organismal viability after different types of DNA damage; 2) the dependence of different tissues on HDR to maintain genome integrity after different types of DNA damage; 3) mutation signatures in tissues of HDR deficient mice caused by different stresses, allowing inference of repair pathway utilized in absence of HDR; 4) how the transcription factor p53 cooperates with HDR in each of these phenotypes; 5) the cooperative role of HDR and NHEJ in DNA repair. We hypothesize that in mice with deletion of the essential HDR gene Brca1, tissues known to develop HDR deficient tumors will have a higher mutation rate and favor p53 mediated programs of arrest over apoptosis compared to non-tumor prone tissues. Specific Aim 1. Determine how HDR preserves viability by mediating tissue specific cellular and transcriptional responses. In wild type or Brca1 deleted mice, we will examine how Brca1/HDR preserves near-term viability after different types of DNA damage by 3 measures: 1) the DNA damage response and cell fate (apoptosis, cell cycle arrest); 2) transcriptomic changes; 3) pathology of different tissues. Specific Aim 2. Determine tissue and DNA damage-specific role of Brca1/HDR in maintaining genome integrity. In this aim, we will examine long-term consequences of HDR deficiency on genome maintenance. Somatic mutations are known to accumulate in normal human tissues with age and are linked to disease. We will examine fidelity of repair and longevity of mice with or without functional HDR that survive different types of DNA damage. Deep sequencing and mutation signature analysis will reveal what tissues rely on HDR and what type of DNA perturbation is most mutagenic in different HDR deficient tissues. Specific Aim 3. Determine viability and stress response in mice deficient in NHEJ or both NHEJ and HDR. Because embryonic deletion of either Brca1 or the NHEJ gene Lig4 (coding for Ligase 4) is lethal, their individual and cooperative roles in adult tissues are not known. In this aim, we will examine survival and stress response after deletion of both HDR and NHEJ pathways in adult mice, leaving only SSA and alt-NHEJ.