SP4: Centrosome integrity as a determinant of replication stress and mitotic dysfunction

中心体在有丝分裂过程中对有丝分裂纺锤体的组装和染色体的忠实分离至关重要。与染色体一样，中心体在细胞周期中只需要复制一次，这意味着相互调节。临床上，中心体成分的胚系突变会导致与复制应激信号缺陷相似的症状，这种情况被称为塞克尔综合征。我们的目的是了解中心体的完整性如何影响DNA复制。初步结果表明，中心体的破坏，通过抑制Polo-like kinase4(Plk4)或中心体成分的耗尽，阻碍了DNA复制分叉的进展。这是通过DNA纤维分析来确定的，该分析监测了核苷酸在单个复制叉中的掺入情况。为了确保复制受损不是异常有丝分裂的结果，我们在中心体成分耗尽之前被CDK4抑制抑制的细胞中进行了这些实验。只有在那时，细胞才被允许进入S阶段，以监测DNA复制。复制分叉进程的减少至少部分是通过激活激酶MK2别名MAPKAPK2来调节的。此外，DNA复制对中心体完整性的依赖主要取决于RAS的激活。总而言之，中心体的破坏激活了MK2，并损害了DNA复制分叉进程。我们现在正计划阐明其潜在的机制及其与有丝分裂的关系。这包括识别由中心体破坏诱导并导致DNA复制受损的信号通路。磷酸化的信号中间产物将与Petra Beli(SP1)合作进行量化。我们将不仅在标准细胞系中检测这一点，还将在SP5 Wollnik提供的塞克尔综合征患者的成纤维细胞中进行检测。此外，我们将与Holger bastians(SP2)合作，测试中心体断裂的这种复制压力是否也会导致纺锤体异常，即在随后的有丝分裂中染色体错误分离。此外，还将与SP-Z一起详细分析因中心体破坏而引起的结构和数字染色体异常。最后，我们将询问中心体异常是否也影响复制不足的DNA在有丝分裂期间如何修复的特征。

Centrosomes are crucial for the assembly of mitotic spindles and faithful separation of chromosomes during mitosis. Like chromosomes, centrosomes need to be duplicated exactly once during the cell cycle, suggesting mutual regulation. Clinically, germ line mutations of centrosomal components lead to similar syndromes as defects in the replication stress signaling, a condition termed Seckel syndrome. Our aim is to understand how centrosomal integrity affects DNA replication. Preliminary results indicate that the disruption of centrosomes, by inhibiting polo-like kinase 4 (PLK4) or by depletion of centrosomal components, hinders the progression of DNA replication forks. This was determined by DNA fiber assays, monitoring the incorporation of nucleotides into single replication forks. To ensure that impaired replication was not a result of aberrant mitoses, we performed these experiments in cells that were arrested by CDK4 inhibition before depleting centrosomal components. Only then, the cells were allowed to enter S phase to monitor DNA replication. Decreased replication fork progression was mediated, at least in part, by activation of the kinase MK2 alias MAPKAPK2. Moreover, the dependence of DNA replication on centrosomal integrity was mostly observed upon Ras activation. In conclusion, the disruption of centrosomes activates MK2 and impairs DNA replication fork progression. We are now planning to elucidate the underlying mechanism and their relation to mitosis. This includes the identification of signaling pathways that are induced by centrosome disruption and that lead to impaired DNA replication. Phosphorylated signaling intermediates will be quantified in cooperation with Petra Beli (SP1). We will assay for this not only in standard cell lines, but also in fibroblasts from patients with Seckel syndrome provided by SP5 Wollnik. Moreover, in cooperation with Holger Bastians (SP2), we will test whether this replication stress upon centrosome disruption also contributes to spindle abnormalities, chromosome missegregation in the subsequent mitosis. Moreover, detailed analyses of structural and numerical chromosome aberrations in response to centrosome disruption will be performed together with SP-Z. Finally, we will ask whether centrosomal abnormalities also affect the characteristics of how under-replicated DNA is repaired during mitosis.