Defining WASp-dependent pathways in replication stress

复制应激反应（RSR）和DNA损伤反应（DDR）的扰动导致 基因组不稳定性复制蛋白A（RPA）是单链DNA（ssDNA）结合蛋白 在RSR和DDR中扮演重要角色。Wiskott-Aldrich综合征的基因组不稳定性 (WAS)，一种原发性免疫缺陷和癌症易感性疾病，但分子 WAS细胞中不稳定基因组的基础仍然未被表征。WASp蛋白 缺乏这种疾病，在细胞质和细胞核中发挥作用。在细胞核中，WASp 功能是防止有害的R环（RNA-DNA杂交体+ ssDNA）的积累， 通过同源定向修复将断裂的DNA末端护送到修复位点的预修复步骤 (HDR)通路因此，WAS患者的淋巴细胞不具备预防和治疗两方面的能力。 解决DNA损伤，提出WAS是一种“遗传毒素敏感”的免疫失调 disorder.我们的基础研究揭示了WASp在RSR中的重要作用， 影响RPA功能下的羟基脲诱导的复制应激，在免疫和 非免疫细胞因此，本提案的总体目标是阐明 WASp如何保障正常复制以及WASp蛋白和途径的详细信息 在复制应激时，与之相关的基因使其能够发挥这种功能。我们将检验这个假设， WASp是预防和管理复制应激和DNA损伤所必需的。具体地说， 我们将在处理阻塞复制分叉的机制中定义WASp角色（在Aim 1中）， 说明WASp如何作用于特定的肌动蛋白状态（G-肌动蛋白与F-肌动蛋白）的改变， RSR（目标2）。实现这些目标将提出WASp作为一种新的RSR因子， WAS是一种RSR功能障碍性疾病，可能为肿瘤发生提供新的机制 在WAS。

Perturbation in the replication-stress response (RSR) and DNA damage response (DDR) causes genomic-instability. Replication protein A (RPA) is a single-strand DNA (ssDNA) binding protein with key roles in the RSR and DDR. Genomic-instability occurs in Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency and cancer susceptibility disorder, yet the molecular underpinnings of unstable genome in WAS cells remain uncharacterized. WASp, the protein deficient in this disorder, functions both in the cytoplasm and nucleus. In the nucleus, WASp functions to prevent the accumulation of harmful R loops (RNA-DNA hybrids + ssDNA) and in the pre-repair step of escorting broken DNA ends to the repair sites by the homology-directed repair (HDR) pathway. Accordingly, WAS patient lymphocytes are poorly-equipped to both prevent and resolve DNA damage, which proposes WAS as a “genotoxin-sensitive” immune dysregulation disorder. Our foundational studies have uncovered an essential role of WASp in the RSR by influencing RPA functions under hydroxyurea-induced replication stress, in both immune and nonimmune cells. Therefore, the overall objective of this proposal is to explicate the molecular details of how WASp safeguards normal replication and which proteins and pathways WASp associates with to enable this function during replicative stress. We will test the hypothesis that WASp is required to both prevent and manage replication stress and DNA damage. Specifically, we will define WASp role in mechanisms that process a blocked replication fork (in Aim 1) and address how WASp role specifically on modifying the actin state (G-actin vs. F-actin) influence RSR (in Aim 2). Achieving these aims will propose WASp as a novel RSR factor, and human WAS as a disease of dysfunctional RSR, which may provide new mechanisms for oncogenesis in WAS.