Profiling DNA damage response following mitotic perturbations.

Ronni S Pedersen,Thorkell Gudjonsson,Jiri Lukas,Rainer Pepperkok,Jean-Karim Hériché,Daniel W Gerlich,Beate Neumann,Jan Ellenberg,Claudia Lukas,Maj-Britt Rask,Gopal Karemore

doi:10.1038/ncomms13887

Abstract

Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells. Unexpectedly, we find that only a subset of these correlations are functionally linked. We identify the genuine mitosis-born DNA damage events and sub-classify them according to penetrance of the observed phenotypes. To demonstrate the potential of this resource, we show that DNA breakage after cytokinesis failure is preceded by replication stress, which mounts during consecutive cell cycles and coincides with decreased proliferation. Together, our results provide a resource to gauge the magnitude and dynamics of DNA breakage associated with mitotic aberrations and suggest that replication stress might limit propagation of cells with abnormal karyotypes.

Highlights

Genome integrity relies on precise coordination between DNA replication and chromosome segregation
Amongst prominent examples of this trait are common fragile sites, which due to paucity of replication origins and topological constraints that obstruct movement of replication forks fail to complete DNA replication in one cell cycle[8]. This generates DNA structures that cannot be detected by cell cycle checkpoints and are transferred to mitosis where they become converted to doublestrand breaks (DSBs) via the MUS81-EME1 nuclease[9]
As a cellular model we used U-2-OS, a human osteosarcoma cell line that has been extensively characterized for DNA-damage response (DDR) including sporadic DSBs generated during cell cycle progression[12]

Summary

Introduction

Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Many RS-initiated and mitosisexecuted DSBs can be repaired already during mitosis[10], a fraction of these lesions is frequently transferred to daughter cells where they become sequestered in 53BP1 nuclear bodies until they are repaired[11,12] Should any of these mechanisms fail, RS-induced and mitosis-propagated DSBs can give rise to structural and numerical chromosome instability, which could in turn fuel cancer progression[13]. To shed light on these issues and generate resource for their further investigation, we systematically silence by siRNA a representative set of established cell cycle regulators, whose disruption impairs major mitotic events[24] By combining this approach with multiparametric profiling of the cell population data, and together with real-time tracking of single cells for several successive generations, we ask whether mitotic errors and DNA breakage in daughter cells are functionally connected, whether RS is involved, and how mitosis-induced DNA-damage response (DDR) affects cell fate decisions

Methods

Results

Conclusion