Structural biology of DNA damage response in chromatin

组蛋白H2 A及其变体H2A.X在细胞对DNA双链断裂的反应中起重要作用 （DSB）在哺乳动物染色质中。在DSB附近，染色质富含H2A.X， 由ATM激酶磷酸化。H2A.X的磷酸化引发了翻译后级联反应， 修饰（PTM），最终将环指E3泛素连接酶RNF 168募集至DNA 损伤部位。RNF 168和同源E2泛素缀合酶UbcH 5c催化单- 组蛋白H2 A和H2A.X在赖氨酸残基13和15处的泛素化（H2 AK 13 ub和H2 AK 15 ub）， 核小体核心颗粒这两个PTM被识别，并确定招聘受损 几种DNA损伤反应（DDR）蛋白的染色质，包括53 BP 1、BRCA 1/RAP 80、RAD 18和 RNF 169控制非同源末端连接（NHEJ）的DSB修复途径之间的平衡 同源重组（HR）。这项研究计划的目标是确定分子 H2 A和H2A.X参与DDR的机制。使用核磁共振光谱，X射线 晶体学，冷冻电子显微镜，生物化学和细胞生物学，我们将探讨如何RNF 168-UbcH 5c 产生H2 AK 13 ub和H2 AK 15 ub，以及这些PTM如何被促进HR的DDR蛋白识别， 核小体的背景。我们还将检验H2A.X在赖氨酸残基处的泛素化 13或15直接有助于染色质解压缩，从而促进DNA修复的募集 蛋白质到DSB。最后，我们将讨论H2 A/H2A.X交换和H2A.X富集的机制。 由人组蛋白伴侣FACT促进的DSB。总的来说，我们的工作将提供基本的 与NHEJ和HR修复机制相关的知识，对人类健康具有重要意义， 在叙述段落中解释。正如研究中经常出现的情况一样，新的未预料到的和 在我们的研究中会出现一些有趣的问题，根据MIRA R35机制，我们将 根据需要解决。

Histone protein H2A and variant H2A.X play central roles in the cellular response to DNA double-strand breaks (DSBs) in mammalian chromatin. Near DSBs, chromatin becomes enriched in H2A.X which gets phosphorylated by ATM kinase. Phosphorylation of H2A.X sets off a cascade of post-translational modifications (PTMs) culminating with the recruitment of RING-finger E3 ubiquitin ligase RNF168 to DNA damage sites. RNF168 and cognate E2 ubiquitin-conjugating enzyme UbcH5c catalyze the mono- ubiquitylation of histones H2A and H2A.X at lysine residues 13 and 15 (H2AK13ub and H2AK15ub) in the nucleosome core particle. These two PTMs are recognized by and determine the recruitment to damaged chromatin of several DNA damage response (DDR) proteins including 53BP1, BRCA1/RAP80, RAD18 and RNF169 that control the balance between the DSB repair pathways of non-homologous end joining (NHEJ) and homologous recombination (HR). The goal of this research program is to determine the molecular mechanisms by which H2A and H2A.X are involved in the DDR. Using NMR spectroscopy, X-ray crystallography, cryo-electron microscopy, biochemistry and cell biology, we will probe how RNF168-UbcH5c generates H2AK13ub and H2AK15ub and how these PTMs are recognized by HR-promoting DDR proteins in the context of the nucleosome. We will also test the hypothesis that ubiquitylation of H2A.X at lysine residues 13 or 15 directly contributes to chromatin decompaction, thereby facilitating the recruitment of DNA repair proteins to DSBs. Finally, we will address the mechanism of H2A/H2A.X exchange and H2A.X enrichment at DSBs promoted by human histone chaperone FACT. Collectively, our work will provide fundamental knowledge relevant to NHEJ and HR repair mechanisms with important implications for human health as explained in the Narrative paragraph. As is often the case in research, it is likely that new unanticipated and fascinating questions will arise during our studies that, in keeping with the MIRA R35 mechanism, we will tackle as needed.