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

中心体对有丝分裂纺锤体的组装和有丝分裂过程中染色体的忠实分离至关重要。像染色体一样，中心体在细胞周期中只需要复制一次，这表明它们是相互调节的。临床上，中心体成分的种系突变导致与复制应激信号缺陷类似的综合征，称为Seckel综合征。我们的目的是了解中心体完整性如何影响DNA复制。初步结果表明，中心体的破坏，通过抑制polo样激酶4 （PLK4）或中心体成分的消耗，阻碍了DNA复制分叉的进展。这是通过DNA纤维测定来确定的，监测核苷酸与单个复制叉的结合。为了确保受损的复制不是异常有丝分裂的结果，我们在耗尽中心体成分之前被CDK4抑制的细胞中进行了这些实验。只有这样，细胞才被允许进入S期以监测DNA复制。减少复制叉的进展至少部分是通过活化激酶MK2别名MAPKAPK2介导的。此外，DNA复制对中心体完整性的依赖主要是在Ras激活时观察到的。总之，中心体的破坏激活了MK2并损害了DNA复制叉的进展。我们现在正计划阐明潜在的机制及其与有丝分裂的关系。这包括鉴定由中心体破坏诱导的信号通路，并导致受损的DNA复制。磷酸化的信号中间体将与Petra Beli （SP1）合作进行量化。我们不仅将在标准细胞系中进行检测，还将在SP5 Wollnik提供的Seckel综合征患者的成纤维细胞中进行检测。此外，在与Holger Bastians （SP2）的合作下，我们将测试这种中心体断裂时的复制胁迫是否也会导致随后有丝分裂中的纺锤体异常、染色体错分离。此外，将与SP-Z一起进行对中心体破坏的结构和数量染色体畸变的详细分析。最后，我们将询问中心体异常是否也会影响有丝分裂期间复制不足的DNA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.