Centrosome impairment causes DNA replication stress through MLK3/MK2 signaling and R-loop formation

Abstract

Running title: Centrosome integrity supports DNA replicationKey words: Centrosome, CEP152, CCP110, SASS6, CEP152, Polo-like kinase 4 (PLK4), DNA replication, DNA fiber assays, R-loops, MLK3, MK2 alias MAPKAPK2, Seckel syndrome, microcephaly. Highlights:• Centrosome defects cause replication stress independent of mitosis.• MLK3, p38 and MK2 (alias MAPKAPK2) are signalling between centrosome defects and DNA replication stress through R-loop formation. • Patient-derived cells with defective centrosomes display replication stress, whereas inhibition of MK2 restores their DNA replication fork progression and proliferation. ABSTRACT Centrosomes function as organizing centers of microtubules and support accurate mitosis in many animal cells. However, it remains to be explored whether and how centrosomes also facilitate the progression through different phases of the cell cycle. Here we show that impairing the composition of centrosomes, by depletion of centrosomal components or by inhibition of polo-like kinase 4 (PLK4), reduces the progression of DNA replication forks. This occurs even when the cell cycle is arrested before damaging the centrosomes, thus excluding mitotic failure as the source of replication stress. Mechanistically, the kinase MLK3 associates with centrosomes. When centrosomes are disintegrated, MLK3 activates the kinases p38 and MK2/MAPKAPK2. Transcription-dependent RNA:DNA hybrids (R-loops) are then causing DNA replication stress. Fibroblasts from patients with microcephalic primordial dwarfism (Seckel syndrome) harbouring defective centrosomes showed replication stress and diminished proliferation, which were each alleviated by inhibition of MK2. Thus, centrosomes not only facilitate mitosis, but their integrity is also supportive in DNA replication. Graphical abstract MK2 p38 ATF2 JUN Conflict 3 BACKGROUND

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“…R-loop staining was performed according to published protocols provided by Zainab Tayeh and Matthias Dobbelstein 22 with a slight modification. Single-cell organoids were seeded and incubated for 3 hours on an 8-well chamber slide (Thermo Fisher Scientific), which was coated previously with 1% Matrigel overnight.…”

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“…Our group demonstrated before that intact centrosomes are necessary for the proper progression of DNA replication forks (Tayeh et al 2020). In other words, disruption of the centrosomes leads to replicative stress and to a subsequent DNA damage response.…”

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