Temporal resolution of phosphorylation-mediated signalling events in DNA Damage Repair

基因组的完整性不断受到内源性和外源性损伤的威胁，导致DNA损伤的产生。这种损伤触发DNA损伤反应（DDR），其中复杂信号网络的激活协调下游事件，所述下游事件可以包括细胞周期停滞、DNA修复、衰老或凋亡。该系统的完整性是至关重要的，因为在许多癌细胞类型中发现的受损DDR网络允许它们绕过这种损伤传感或逃避诱导凋亡。DDR途径包括检测损伤的传感器分子、放大信号的信号转导子和被募集/激活以介导下游事件的效应分子。不同的网络被激活取决于损伤的类型和发生损伤的细胞周期阶段。长期以来，可逆磷酸化一直被认为是DDR中的关键调节事件，但尽管对介导信号传导事件的主要Ser/Thr激酶了解很多，但令人惊讶的是，对其主要对应物（包括Ser/Thr磷酸酶PP 1和PP 2A）的同样重要的作用知之甚少。 PP 1-和PP 2A-介导的去磷酸化事件的分析是复杂的，其独特的调节机制，从而一个共同的催化亚基之间的共享池的调节亚基动态分布，形成具有特定的定位和底物特异性的全酶复合物。我们已经开发出的策略，使我们能够监测全酶复合物之间的催化亚基的动态亚细胞分布，并改变具有高特异性的单个复合物的水平或活性。我们的荧光成像与定量相互作用组映射相结合，武装了我们与核PP 1分布的全面的空间地图，并确定了几个复合物直接向DDR基板。标准工作流程包括互补的定量亲和纯化/质谱（AP/MS）和邻近标记方法，以高分辨率映射时空相互作用组，基于CRISPR/Cas9的诱变和定量磷酸蛋白质组学映射，以鉴定底物中靶向的特定残基。 为了更全面地了解这一复杂途径的信号动力学，我们有了以下三个短期目标，可以从功能上详细分析PP 1和PP 2A磷酸酶复合物对DDR的贡献： 1.在不同类型的DDR反应中，核全酶复合物之间PP 1和PP 2A催化亚基的稳态分布的地图变化。 2.组装损伤后早期、中期和晚期时间点DDR靶向PP 1和PP 2A全酶复合物的时空相互作用组的网络图。 3.评估特定复合物的破坏对损伤检测、途径选择、修复时机和DDR相关去磷酸化事件的影响

The integrity of the genome is constantly threatened by endogenous and exogenous insults that lead to generation of DNA damage. This damage triggers a DNA damage response (DDR) in which activation of a complex signaling network coordinates downstream events that can include cell cycle arrest, DNA repair, senescence or apoptosis. Integrity of this system is critical, as compromised DDR networks found in many cancer cell types allow them to either bypass this damage sensing or evade induction of apoptosis. The DDR pathway comprises sensor molecules that detect the damage, signal transducers that amplify the signal and effector molecules that are recruited/activated to mediate downstream events. Distinct networks are activated depending on the type of damage and cell cycle stage at which it occurs. Reversible phosphorylation has long been known to be a key regulatory event in DDR, but although much is known about the principle Ser/Thr kinases that mediate signalling events, surprisingly little is known about the equally essential roles of their primary counterparts, which include the Ser/Thr phosphatases PP1 and PP2A. Analysis of PP1- and PP2A-mediated dephosphorylation events is complicated by their unique regulatory mechanism, whereby a common catalytic subunit is dynamically distributed between a shared pool of regulatory subunits to form holoenzyme complexes with specific localizations and substrate specificities. We have developed strategies that allow us to monitor the dynamic subcellular distribution of catalytic subunits between holoenzyme complexes and alter the level or activity of individual complexes with high specificity. Our combination of fluorescence imaging with quantitative interactome mapping has armed us with a comprehensive spatial map of nuclear PP1 distribution and identified several complexes directed toward DDR substrates. Standard workflows include complementary quantitative affinity purification/mass spectrometry (AP/MS) and proximity labeling approaches to map spatiotemporal interactomes with high resolution, CRISPR/Cas9-based mutagenesis and quantitative phosphoproteomic mapping to identify the specific residues targeted in substrates. With a long-term goal of building a more complete picture of the signalling dynamics of this complex pathway, the following three short-term objectives will allow us to functionally dissect the contribution of PP1 and PP2A phosphatase complexes to DDR in unprecedented detail: 1. Map changes in the steady-state distribution of PP1 and PP2A catalytic subunits between nuclear holoenzyme complexes in response to distinct type of DDR. 2. Assemble a network map of the spatiotemporal interactomes of DDR-targeted PP1 and PP2A holoenzyme complexes at early, mid and late time points following damage. 3. Assess the effects of disruption of specific complexes on damage detection, pathway choice, repair timing and DDR-associated dephosphorylation events