Ki-67 is necessary during DNA replication for fork protection and genome stability.

BACKGROUND: The proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years, but investigations on its biological functions have lagged. Recently, Ki-67 has been shown to regulate both the composition of the chromosome periphery and chromosome behaviour in mitosis as well as to play a role in heterochromatin organisation and gene transcription. However, how the different roles for Ki-67 across the cell cycle are regulated and coordinated remain poorly understood. The progress towards understanding Ki-67 function have been limited by the tools available to deplete the protein, coupled to its abundance and fluctuation during the cell cycle. RESULTS: Here, we use a doxycycline-inducible E3 ligase together with an auxin-inducible degron tag to achieve a rapid, acute and homogeneous degradation of Ki-67 in HCT116 cells. This system, coupled with APEX2 proteomics and phospho-proteomics approaches, allows us to show that Ki-67 plays a role during DNA replication. In its absence, DNA replication is severely delayed, the replication machinery is unloaded, causing DNA damage that is not sensed by the canonical pathways and dependent on HUWE1 ligase. This leads to defects in replication and sister chromatids cohesion, but it also triggers an interferon response mediated by the cGAS/STING pathway in all the cell lines tested. CONCLUSIONS: We unveil a new function of Ki-67 in DNA replication and genome maintenance that is independent of its previously known role in mitosis and gene regulation.

A dual system to manipulate protein levels for DNA replication- and cell cycle-related studies.

Investigation of cell cycle-regulated processes often necessitates the rapid manipulation of individual protein levels in synchronized populations over the course of a cell cycle. In the budding yeast, the two major orthogonal approaches by which this is accomplished are conditional gene expression via inducible or repressible promoters and regulated metabolic destabilization of a protein of interest via ubiquitin-mediated degradation signals. Here, we describe an application of these principles to the investigation of DNA damage signaling during replication. Using a combination of conditional gene expression via a tetracycline-repressible promoter and inducible protein degradation via an auxin-regulated degron system, we have analyzed the cross talk between factors controlling the bypass of lesions during replication and the activation of the DNA damage checkpoint. Here, we describe the basic principles underlying our experimental system and provide a detailed protocol to analyze the cross talk between damage bypass and checkpoint signaling. Finally, we discuss the advantages and disadvantages of using this approach and point out possible applications to other regulatory events and other organisms.