Post-translational Modifications in DNA Damage Response: a Structural Perspective

我们的长期目标是帮助破译细胞如何处理 DNA 损伤以维持基因组完整性。 DNA 损伤反应 (DDR) 涉及感知 DNA 损伤并协调细胞周期进程以及 DNA 修复、细胞凋亡和 DNA 损伤耐受途径的蛋白质复合物。组蛋白和非组蛋白的可逆翻译后修饰 (PTM) 对于 DDR 机制在 DNA 损伤位点的组装至关重要，但人们对其机制知之甚少。我们提出的研究重点是从结构角度探讨 PTM 如何参与 DDR 中时间和空间依赖性蛋白质关联的控制。具体来说，我们将研究赖氨酸甲基化和泛素化在 DNA 双链断裂 (DSB) 修复和跨损伤 DNA 合成 (TLS) 的 DNA 损伤耐受中的作用。 53BP1 是通过非同源末端连接修复 DNA 的重要介质。为了响应 DSB，53BP1 与在赖氨酸 20 处二甲基化的组蛋白 H4 (H4K20me2) 结合，在没有损伤的情况下，它被认为与赖氨酸脱甲基酶 JMJD2A 复合。 DSB 诱导后，一些泛素连接酶（例如 RNF8）被招募到损伤位点，在这些位点泛素化组蛋白和 JMJD2A。 JMJD2A 的泛素化会触发其降解，促进 53BP1 与 H4K20me2 相互作用并重新定位到 DSB。在目标 1 下，我们将探讨 53BP1、JMJD2A 和结构相关蛋白 PHF20 的招募机制。我们将表征这些蛋白质与核小体核心颗粒的相互作用。在目标 2 下，我们将研究 DNA 钳 PCNA 响应 DNA 损伤的单泛素化如何通过 TLS 激活损伤耐受性。我们将探讨 PCNA 泛素化如何激活 TLS DNA 聚合酶 Rev1 和 TLS。我们的假设是泛素化改变了 PCNA-Rev1 相互作用，并作为打开 TLS 的双开关发挥作用。我们的研究将成为设计体内检测的基础，从而提高我们对 DDR 的了解。 H4K20 甲基化的改变是人类肿瘤细胞的标志，多种 DDR 蛋白的突变与人类癌症易感性有关。由于 53BP1 的缺失会逆转 BRCA1 突变细胞的癌症表型，因此抑制 53BP1 募集到 DSB 位点可能具有癌症治疗的潜力。 Rev1 和 TLS 有助于癌细胞获得治疗耐药性。

Our long-term goal is to help decipher how cells process DNA lesions in the maintenance of genomic integrity. The DNA damage response (DDR) involves protein complexes that sense DNA lesions and coordinate cell cycle progression along with DNA repair, apoptosis and DNA damage tolerance pathways. Reversible post-translational modifications (PTMs) of histone and non-histone proteins are essential for the DDR machinery to assemble at DNA damage sites but the mechanisms are poorly understood. Our proposed research focuses on probing, from a structural perspective, how PTMs participate in the control of time- and space-dependent protein associations in the DDR. Specifically, we will investigate the roles of lysine methylation and ubiquitylation in the repair of DNA double-strand breaks (DSBs) and in DNA damage tolerance by translesion DNA synthesis (TLS). 53BP1 is an important mediator of DNA repair by non-homologous end joining. In response to DSBs, 53BP1 associates with histone H4 dimethylated at lysine 20 (H4K20me2), which, in the absence of damage, is thought to be complexed to the lysine demethylase JMJD2A. Upon DSB induction, several ubiquitin ligases such as RNF8 are recruited to damage sites where they ubiquitylate histones and JMJD2A. The ubiquitylation of JMJD2A triggers its degradation, facilitating 53BP1 interaction with H4K20me2 and relocalization to DSBs. Under Aim 1, we will probe the recruitment mechanisms of 53BP1, JMJD2A and structurally related protein PHF20. We will characterize interactions of these proteins with the nucleosome core particle. Under Aim 2, we will investigate how mono-ubiquitylation of the DNA clamp PCNA in response to DNA damage activates damage tolerance by TLS. We will probe how the TLS DNA polymerase Rev1 and TLS are activated by PCNA ubiquitylation. Our hypothesis is that ubiquitylation alters PCNA–Rev1 interaction and functions as a double-switch to turn on TLS. Our studies will be the basis for designing in vivo assays that can elevate our knowledge of the DDR. Alteration of H4K20 methylation is a hallmark of human tumor cells and mutations in several DDR proteins are linked to cancer predispositions in humans. Because loss of 53BP1 reverses cancer phenotypes of BRCA1 mutant cells, inhibition of 53BP1 recruitment to DSB sites could have potential for cancer therapy. Rev1 and TLS contribute to the acquired therapeutic resistance of cancer cells.