THE ROLE OF FAN1 NUCLEASE IN KIDNEY AND DIGESTIVE SYSTEM HEALTH AND FUNCTION

项目摘要 DNA损伤的忠实修复是维持基因组稳定的关键。缺乏修复或不适当的DNA 修复可能导致突变、细胞生长失控、细胞死亡或生长停滞。我们已确定风扇1 核酸酶作为维持基因组稳定性的潜在效应器。FAN1与链间病毒有牵连 作为一种核酸酶的交联链(ICL)修复，负责解钩和释放交联或受损的DNA 基地。ICL修复蛋白的突变通常会导致骨髓衰竭或癌症；然而， FAN1可引起一种慢性肾脏疾病，称为巨细胞性间质性肾炎(KIN)。亲属患者 最终发展为需要透析或肾移植的肾功能衰竭。潜在的机制是 目前尚不清楚FAN1突变是如何导致亲缘关系的。这可能是肾功能障碍通过 内源性产生的ICL的修复缺陷，或者可能是由于FAN1在 规范的ICL修复。最近，有研究表明，FAN1的突变使人更容易患上 结直肠癌和胰腺癌，是三核苷酸重复序列扩张性疾病的基因修饰物，如 亨廷顿氏症。综上所述，这些发现表明FAN1可能具有外部功能 规范的ICL修复。在拟议的研究中，我们的目标是弄清FAN1的各种分子机制 在体内的作用以及确定驱动亲缘关系的致病内源性损害。在目标1中，目标是使用 一种多管齐下的方法确定了FAN1在体细胞重复不稳定中的作用。使用鼠标模型 三核苷酸重复扩增我们将评估Fan1的缺失是否会驱动组织中的重复扩增- 特定的方式或通过生殖系遗传。我们还将确定FAN1是否会影响微卫星重复 不稳定(MSI)，结肠癌的一种常见表型。在目标2中，我们将确定 FAN1在DNA复制中的作用，亲缘关系中多倍化的机制，以及肿瘤发生的易感性。 我们将使用Fan1基因敲除小鼠模型来概括人类亲属的表型。通过将细胞从 该模型和在存在/不存在DNA复制应激的情况下执行各种DNA复制分析 或损害，我们将阐明Fan1在体内的作用。我们还将进行实验，以更好地 了解FAN1在亲缘关系和肿瘤发生中的作用。最后，在目标3中，我们将确定致病病变(S) 在没有风扇1的情况下驾驶亲人。这项提案的发现有可能揭示 FAN1如何抑制肾脏和消化系统功能障碍的基本原则。

Project Summary The faithful repair of DNA damage is critical to maintain genome stability. Lack of repair or inappropriate DNA repair can lead to mutations, uncontrolled cell growth, cell death, or growth arrest. We have identified FAN1 nuclease as a potential effector for maintaining genome stability. FAN1 has been implicated in interstrand crosslink (ICL) repair as a nuclease responsible for unhooking and releasing the crosslinked or damaged DNA base. Mutations in ICL repair proteins usually give rise to bone marrow failure or cancer; however, mutations in FAN1 cause a form of chronic kidney disease termed Karyomegalic Interstitial Nephritis (KIN). KIN patients ultimately develop renal failure requiring dialysis or kidney transplantation. The underlying mechanism as to how FAN1 mutations gives rise to KIN still remains unknown. It may be that kidney dysfunction occurs through defective repair of endogenously produced ICLs or it could be due to independent functions of FAN1 outside of canonical ICL repair. More recently, it was shown that mutations in FAN1 confer a greater susceptibility to colorectal and pancreatic cancer, and are genetic modifiers for trinucleotide repeat expansion diseases, such as Huntington's disease. Together, these findings suggest that FAN1 may have functions outside of the canonical ICL repair. In the proposed research, we aim to discern the various molecular mechanisms of FAN1 function in vivo as well as to identify the causative endogenous lesions driving KIN. In Aim 1, the goal is to use a multi-pronged approach determine the role of FAN1 in somatic repeat instability. Using a mouse model of trinucleotide repeat expansion we will assess if the absence of Fan1 drives repeat expansion in a tissue- specific manner or through germline inheritance. We will also determine if FAN1 affects microsatellite repeat instability (MSI), a common phenotype observed in colon cancer. In Aim 2, we will determine the function of FAN1 in DNA replication, the mechanism of polyploidization in KIN, and the predisposition to tumorigenesis. We will use a Fan1 knockout mouse model that recapitulates the human KIN phenotype. By isolating cells from this model and performing various DNA replication assays in the presence/absence of DNA replication stress or damage, we will shed light on the in vivo role for Fan1. We will also perform experiments to better understand the role for FAN1 in KIN and tumorigenesis. Finally, in Aim 3 we will identify the causative lesion(s) driving KIN in the absence of FAN1. The findings from this proposal have the potential to uncover the fundamental principles of how FAN1 acts to suppress dysfunction of the kidney and digestive system.