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，以保持基因组的完整性。DNA损伤反应(DDR)涉及感知DNA损伤并协调细胞周期进展的蛋白质复合体，以及DNA修复、细胞凋亡和DNA损伤耐受途径。组蛋白和非组蛋白的可逆翻译后修饰(PTM)对于DDR机制在DNA损伤部位组装是必不可少的，但其机制尚不清楚。我们建议的研究集中在从结构的角度探索PTMS如何参与DDR中依赖时间和空间的蛋白质关联的控制。具体地说，我们将研究赖氨酸甲基化和泛素化在DNA双链断裂(DSB)修复和跨病变DNA合成(TLS)对DNA损伤耐受中的作用。53BP1是DNA非同源末端连接修复的重要介导物。作为对DSB的反应，53BP1与赖氨酸20位的组蛋白H4(H4K20me2)发生二甲基化，在没有损伤的情况下，组蛋白H4被认为与赖氨酸去甲基酶JMJD2A络合。在DSB诱导后，几种泛素连接酶如RNF8被招募到损伤位置，在那里它们泛素化组蛋白和JMJD2A。JMJD2A的泛素化引发其降解，促进53BP1与H4K20me2的相互作用并重新定位为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。我们将描述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钳增殖细胞核抗原在DNA损伤时的单一泛素化如何通过TLS激活损伤耐受。我们将探讨TLS DNA聚合酶Rev1和TLS是如何被增殖细胞核抗原泛素化激活的。我们的假设是，泛素化改变了PCNA-Rev1的相互作用，并作为启动TLS的双开关发挥作用。我们的研究将成为设计体内试验的基础，从而提高我们对DDR的认识。H4K20甲基化的改变是人类肿瘤细胞的一个标志，几种DDR蛋白的突变与人类的癌症易感性有关。由于53BP1的丢失可以逆转BRCA1突变细胞的癌症表型，因此抑制53BP1向DSB位点的募集可能具有癌症治疗的潜力。Rev1和TLS参与了癌细胞的获得性治疗耐药。

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.