Phase separation in PML nuclear bodies through a multivalent polySUMO-2/3 network is required for efficient DNA recombination and repair

生物化学活动的空间组织对每个活细胞来说都是一项极其重要的任务。哺乳动物细胞通过将基本细胞功能集中到膜封闭的隔室或无膜细胞器中来完成这项任务。无膜细胞器的形成、维持和功能的分子机制现在才开始被揭开。无膜细胞器组装的主要物理驱动力是液-液相分离（LLPS），并且该机制由具有特定生物物理性质的特定类别的分子（蛋白质、RNA、DNA）启动。哺乳动物细胞核中的无膜细胞器包括核仁、Cajal小体和所谓的早幼粒细胞白血病核小体（PML NB）。虽然PML NB的生化功能尚未阐明，但这些核结构域已被认为通过LLPS机制组装和发挥功能，但缺乏体内证据。通过这项研究计划，我们希望分析在同源定向DNA重组和修复与NB物理相关的染色质过程中PML体内潜在的相分离特征。为此，我们将结合联合收割机生物化学和生物物理体外研究与先进的活细胞和超分辨率成像技术和计算机建模。这些分析将证明我们的假设，即PML核体作为专用的“微反应器”，其中通过相分离将特异性DNA重组和修复因子浓缩到活性PML NB中来支持同源DNA重组。

The spatial organization of biochemical activities is an extremely important task for each living cell. Mammalian cells manage this task by concentrating essential cellular functions into membrane-enclosed compartments or into membrane-less organelles. The molecular mechanisms underlying the formation, the maintenance and the function of membrane-less organelles is only now begining to be unraveled. The main physical driver of the assembly of membrane-less organelles is liquid-liquid phase seperation (LLPS) and this mechanism is initiated by a specific class of molecules (Protein, RNA, DNA) with particular biophysical properies. Membrane-less organelles in the nucleus of mammalian cells include the nucleolus, Cajal bodies and the so-called promyelocytic leukemia nuclear bodies (PML NBs). While the biochemical function of PML NBs is not yet elucidated these nuclear domains have been suggested to assemble and function via LLPS mechanisms, but in vivo evidence for this is lacking. With this research proposal we want to analyze the potential phase seperation features within PML bodies during homology-directed DNA recombination and repair of chromatin physically associated with the NBs. To this end we will combine biochemical and biophysical in vitro studies with advanced live-cell and super-resolution imaging technologies and computer modeling. These analyses shall prove our hypothesis that PML nuclear bodies serve as dedicated 'micro-reactors' where homologous DNA recombination is supported by concentrating specific DNA recombination and repair factors into active PML NBs by phase seperation.