Multiscale proteomics studies of DNA repair and genomic stability

基因组的完整性不断受到遗传毒性物质和复制压力的挑战。损失 基因组的稳定性可能会导致癌症、过早衰老和神经退化等病理情况。 在基因组改变时，细胞协调一个统称为DNA的分子通路网络 损伤反应(DDR)，发出信号并促进DNA损伤的修复，阻止细胞周期进展，直到 基因组的完整性得到了恢复。有几条证据表明，基因组的不稳定有助于 肿瘤发生、癌症进展和治疗耐药的发展。基因分析已经确定 参与DNA损伤反应的高、中、低外显性癌症易感基因 并进行修复。尽管多年来已经进行了许多机制和遗传学研究，但系统的 对不同类型病毒反应过程中染色体蛋白质变化的分析 基因组微扰仍然是欠缺的。 基于高分辨率质谱学的蛋白质组学是一种可靠的鉴定和鉴定 对复杂混合物中的蛋白质进行定量。亲和纯化与质谱联用 实验导致了复杂的蛋白质相互作用网络的发现，蛋白质复合体的分析 在染色质上组装更具挑战性，因为纯化方法效率低下， 与质谱学有偏差或不兼容。然而，由于技术上的原因，这种情况正在迅速演变 蛋白质组学研究进展。 我建议对蛋白质景观进行全面和公正的定量和动力学分析 在对不同的遗传毒性物质的反应中，组装在完全功能的核和染色体上，一个 我称之为多尺度蛋白质组学的方法。具体地说，我将使用从 脊椎动物非洲爪哇卵与最先进的质谱分析相结合。这是无细胞的 系统允许在细胞系统中无法实现的实验操作，并允许前所未有的 DNA损伤反应蛋白质组的特征。我假设特定的蛋白质亚群 在不同的损伤条件下被招募到染色质不仅决定了对DNA损伤的反应，而且 还有冗余修复途径的使用，这应该会对诱变剂的发生有一些帮助 在癌症基因组中发现的修复形式。与戈蒂埃实验室的其他成员一起，我将验证 并对非洲爪哇提取物和人类细胞中的发现进行了功能表征。 了解响应和修复DNA损伤的蛋白质网络是如何工作的 有可能影响人类健康。识别有用的合成致命相互作用，这可能会增强 化疗药物对提高实验性基因治疗的安全性和适用性的有效性。因此， 我们预计我们的研究将为DNA损伤反应的调控提供新的见解， 有助于更好地理解细胞如何在遗传毒性作用下保持其基因组的稳定性 侮辱。 好了！

The integrity of the genome is continuously challenged by genotoxic agents and replication stress. Loss of genome stability can lead to pathological conditions such as cancer, premature aging, and neurodegeneration. Upon genomic alterations, cells coordinate a network of molecular pathways collectively known as the DNA damage response (DDR) that signals and promotes repair of DNA lesions, halting cell cycle progression until genome integrity is restored. Several lines of evidence indicate that genomic instability contributes to oncogenesis, cancer progression, and development of therapy resistance. Genetic analyses have identified high-, moderate-, and low-penetrance cancer susceptibility genes that are involved in DNA damage response and repair. Although many mechanistic and genetic studies have been performed over the years, a systematic analysis of the protein changes taking place on the chromosomes during the response to different types of genome perturbations is still lacking. High-resolution mass spectrometry-based proteomics is a robust method for the identification and quantification of proteins from complex mixtures. While affinity purification combined with mass spectrometry experiments have led to the discovery of intricate protein interaction networks, analyses of protein complexes assembled on chromatin have been much more challenging because purification methods are inefficient, biased or not compatible with mass spectrometry. However, this is rapidly evolving due to technological advances in proteomics. I propose to perform a comprehensive and unbiased quantitative and kinetic analysis of the protein landscapes assembled on fully functional nuclei and chromosomes during the response to different genotoxic agents, an approach that I call multiscale proteomics. Specifically, I will employ the cell-free extracts derived from the vertebrate Xenopus laevis eggs combined with state-of-the-art mass spectrometry analyses. This cell-free system allows experimental manipulations that cannot be achieved in cell systems and permits unprecedented characterization of the DNA damage response proteomes. I hypothesize that specific subsets of proteins recruited to chromatin under different damage conditions dictate not only the response to DNA damage, but also the usage of redundant repair pathways, which should shed some light on the occurrence of mutagenic forms of repair found in cancer genomes. Together with other members of the Gautier laboratory, I will validate and functionally characterize the findings in both Xenopus extracts and in human cells. Understanding how the protein networks that respond to and repair DNA damage work holds considerable potential to impact human health. From identifying useful synthetic lethal interactions that might enhance the efficacy of chemotherapy drugs to improving the safety and applicability of experimental gene therapies. Thus, we anticipate our studies will provide new insights on the regulation of the DNA damage responses, contributing to a better understanding of how the cells maintain the stability of their genomes upon genotoxic insults. !