Centrioles are central structural elements of centrosomes and cilia. In human cells daughter centrioles are assembled adjacent to existing centrioles in S-phase and reach their full functionality with the formation of distal and subdistal appendages one-and-a-half cell cycle later, as they exit their second mitosis. Current models postulate that the centriolar protein centrobin acts as placeholder for distal appendage proteins that must be removed to complete distal appendage formation. Here, we investigated in non-transformed human epithelial RPE1 cells the mechanisms controlling centrobin removal and its effect on distal appendage formation. Our data are consistent with a speculative model in which centrobin is removed from older centrioles due to a higher affinity for the newly born daughter centrioles, under the control of the centrosomal kinase Plk1. This removal also depends on the presence of subdistal appendage proteins on the oldest centriole. Removing centrobin, however, is not required for the recruitment of distal appendage proteins, even though this process is equally dependent on Plk1. We conclude that Plk1 kinase regulates centrobin removal and distal appendage formation during centriole maturation via separate pathways.

A tight synchrony between the DNA and centrosome cycle is essential for genomic integrity. Centriole disengagement, which licenses centrosomes for duplication, occurs normally during mitotic exit. We recently demonstrated that mild DNA replication stress in untransformed human cells causes premature centriole disengagement at mitotic entry, leading to transient multipolar spindles that favour chromosome mis-segregation. How mild replication stress accelerate the centrosome cycle at the molecular level remained, however, unclear. Using expansion microscopy, we show that mild replication stress already induces premature centriole disengagement in G2 via the ATR-Chk1 axis of the DNA damage repair pathway. We demonstrate that this results in a subcritical Plk1 kinase activity that is insufficient for rapid mitotic entry. Nevertheless, it primes the pericentriolar matrix for Separase-dependent disassembly causing premature centriole disengagement in G2. We postulate that the differential requirement of Plk1 activity in the DNA and centrosome cycles explains how mild replication stress disrupts the synchrony between both processes and contributes to genomic instability.