A239: Investigating the role of PKC epsilon in the response to DNA catenation at the metaphase-to-anaphase boundary.

Joanna Kelly1,Tanya Pike1,Nicola Brownlow1,3,Peter Parker1,2

1The Francis Crick Institute, London, UK,2King’s College London, London, UK,3Center for Genomic Regulation, Barcelona, Spain

Presenting date: Monday 2 November
Presenting time: 12.20-13.10

Background

The cell cycle is tightly regulated to safeguard the complete and equal division of the nuclear contents between the daughter cells. DNA catenation generated during replication must be fully resolved for accurate segregation to occur. PKC epsilon (PKC?) has been shown to play a crucial role in mediating a catenation-induced delay at the metaphase to anaphase transition. PKC? promotes a metaphase delay in the presence of unresolved DNA catenation to avoid damage to the genome1. PKC? cleavage, at two distinct sites, has been observed during mitosis. We wanted to investigate if cleavage at either of these sites is functionally significant for induction of the metaphase delay.

 

Method

A live cell time-lapse imaging approach was used to assess the robustness of the metaphase catenation response in DLD1 cells expressing inducible GPF-tagged PKC? containing point mutations to block cleavage. The time spent in metaphase was measured in the presence or absence of treatment with ICRF-193, which generates persistent catenation by blocking topoisomerase II activity.

Results

PKC? cleavage is required for the catenation-induced delay during the transition from metaphase to anaphase.  Cells expressing wild type PKC? demonstrate the characteristic catenation-induced delay at the metaphase-to-anaphase transition. When PKC epsilon cannot be cleaved we observed a reduction in this delay indicating a failure to respond to the presence of excess catenation.

Conclusion

PKC? cleavage during mitosis appears to be important to the implementation of the catenation-induced metaphase delay. Understanding the regulation of PKC?

and this cleavage will provide important insight into the role of this kinase in the cellular response to catenation.

Additionally, due to a reliance on this PKC? for this response in transformed cells, identifying the pathways involved in its regulation may offer a potentially novel target for cancer therapeutics.