Hochschulschrift:
Dissertation, Albert-Ludwigs-Universität Freiburg im Breisgau, 2018
Anmerkungen:
Beschreibung:
Abstract: Introduction: Progression through the cell cycle is tightly regulated by different cyclin-dependent kinases and their activating cyclin subunits. Stage-specific proteolysis of cyclins and other cell cycle regulators is important for transition to the next cell cycle phase. The anaphase-promoting complex/cyclosome (APC/C) is an E3-ubiquitin ligase that controls mitosis and G1 through degradation of these proteins. Through its activating subunits Cdh1 and Cdc20 the APC/C assures substrate-specifity. While Cdc20 regulates progression through mitosis, Cdh1 is activated in late mitosis to coordinate accurate entry into S-phase. Thereby, the APC/C is crucial for maintaining genomic stability during the cell cycle.<br>Results: Characterization of a Cdh1-kd revealed strong stabilization of the substrates cyclin A/B leading to diminished loading of mini-chromosome maintenance (MCM) proteins on replication origins in G1. Stabilization of cyclin A/B may cause the observed premature entry into S-phase, while the reduced loading of MCMs in G1 could be responsible for the prolonged replication in S-phase seen in Cdh1-kd cells. Plk1 was stabilized in Cdh1-kd cells, which may cause bypass of the Cdh1-Plk1 dependent DNA damage checkpoint. Indeed, potential replication stress in Cdh1-kd cells did not lead to G2/M arrest, but was enforced by inhibition of the Cdh1 substrate Plk1. Underreplicated DNA and replication intermediates in mitosis may be the reason for increased genomic instability, namely lagging chromosomes, anaphase bridges and micronuclei in Cdh1-kd cells detected by live-cell imaging. Monitoring of 53BP1, a DNA-repair marker, in living cells could also prove amplified DNA-damage through increased double-strand breaks in Cdh1-kd cells. In addition, aberrant cytokinesis and the development of tetraploid cells generated by misseparation of chromosomes during mitosis were enhanced in Cdh1-kd cells.<br>Conclusions: Downregulation of the tumor suppressor APC/C-Cdh1 leads to deregulation of DNA-replication by stabilizing cyclin A and B in G1 and reduced loading of replication origins with MCM proteins resulting in enhanced genomic instability