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

项目摘要/摘要 这个项目的长期目标是描述维持生存能力的途径和机制。 以及暴露在环境遗传毒性应激下的成年哺乳动物的基因组完整性。DNA损伤可能是 通过高保真通路同源定向修复(HDR)或通过容易出错的非同源末端修复 加入(NHEJ)。成年脊椎动物HDR缺失的后果还不是很清楚，因为生殖系 基因敲除是胚胎致死的，因此，我们的大部分认识都来自于细胞培养模型或推断 由HDR缺乏症引起的肿瘤。 为了满足这一关键需求，我们培育出了可以进行诱导缺失的基因工程小鼠 在发育后的每个组织中，高达99%的必需HDR基因BRCA1。我们将使用此模型来 解决以下关键问题：1)不同类型的DNA后，HDR在细胞/组织/生物体活性中的作用 损伤；2)不同组织对HDR的依赖，以维持不同类型DNA后的基因组完整性 损伤；3)不同应激引起的HDR缺陷小鼠组织中的突变特征，允许推断 在缺乏HDR的情况下利用的修复途径；4)转录因子P53如何与HDR在各自的 5)HDR和NHEJ在DNA修复中的协同作用。我们假设在小鼠身上 如果必要的HDR基因BRCA1缺失，已知的发生HDR缺陷肿瘤的组织将有更高的 与不容易发生肿瘤的组织相比，突变率和更倾向于P53介导的停滞程序而不是凋亡。 具体目标1.确定HDR如何通过调节组织特异性细胞和 转录反应。在野生型或BRCA1缺失的小鼠中，我们将研究BRCA1/HDR如何保存 不同类型DNA损伤后的近期存活率通过3个指标：1)DNA损伤反应和细胞 FATE(细胞凋亡，细胞周期停滞)；2)转录改变；3)不同组织的病理学。 特定目标2.确定BRCA1/HDR在维持基因组中的组织和DNA损伤特异性作用 正直。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。 众所周知，随着年龄的增长，体细胞突变会在正常人体组织中积累，并与疾病有关。我们 将检测具有或不具有功能性HDR的小鼠在不同类型的 DNA损伤。深度测序和突变特征分析将揭示哪些组织依赖HDR，哪些组织依赖HDR 在不同的HDR缺陷组织中，DNA扰动的类型是最具突变性的。 具体目标3.测定NHEJ缺乏或NHEJ和HDR同时缺乏的小鼠的生存能力和应激反应。 因为BRCA1或NHEJ基因Lig4(编码Ligase 4)的胚胎缺失是致命的，他们的个体 成人组织中的合作角色尚不清楚。在这个目标中，我们将研究生存和压力反应。 在成年小鼠的HDR和NHEJ通路都缺失后，只剩下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.