Centrosome integrity as a determinant of replication stress and mitotic dysfunction

Matthias Dobbelstein, Göttingen


Centrosomes are crucial in the assembly of mitotic spindles and faithful separation of chromosomes in 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 Seckel patients (in collaboration with Bernd Wollnik, SP5). 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.

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