Systems Modelling of NHEJ Reveals the Importance of Redox Regulation of Ku70/80 in the Dynamics of DNA Damage Foci

David Dolan,Daryl Shanley,Graham Smith,Glyn Nelson,Anze Zupanic,David C Samuels

doi:10.1371/journal.pone.0055190

Abstract

The presence of DNA double-stranded breaks in a mammalian cell typically activates the Non-Homologous End Joining (NHEJ) pathway to repair the damage and signal to downstream systems that govern cellular decisions such as apoptosis or senescence. The signalling system also stimulates effects such as the generation of reactive oxygen species (ROS) which in turn feed back into the damage response. Although the overall process of NHEJ is well documented, we know little of the dynamics and how the system operates as a whole. We have developed a computational model which includes DNA Protein Kinase (DNA-PK) dependent NHEJ (D-NHEJ) and back-up NHEJ mechanisms (B-NHEJ) and use it to explain the dynamic response to damage induced by different levels of gamma irradiation in human fibroblasts. Our work suggests that the observed shift from fast to slow repair of DNA damage foci at higher levels of damage cannot be explained solely by inherent stochasticity in the NHEJ system. Instead, our model highlights the importance of Ku oxidation which leads to increased Ku dissociation rates from DNA damage foci and shifts repair in favour of the less efficient B-NHEJ system.

Highlights

DNA Double-strand breaks (DSB), arguably the most dangerous kind of DNA damage, are caused by reactive oxygen species (ROS) which are produced as a by-product of cellular respiration as well as various environmental stresses
Non-Homologous End Joining (NHEJ) uses two competing pathways: the faster and more accurate repair pathway, DNA Protein Kinase (DNA-PK) Dependent NHEJ (D-NHEJ), mediated by Ku, DNA-PKcs and Ligase IV [3] (Figure 1A); and the recently identified slower, more inaccurate Backup NHEJ system (B-NHEJ) [4,5] mediated by Poly [ADP-ribose] polymerase 1 (PARP-1) and Ligase III (Figure 1B), which are better known as key components of single strand DNA break repair [6]
When a cell is in an unstressed state, damage foci still form indicating that a cell undergoes some damage when at rest in its typical environment (Figure 3)

Summary

Introduction

DNA Double-strand breaks (DSB), arguably the most dangerous kind of DNA damage, are caused by reactive oxygen species (ROS) which are produced as a by-product of cellular respiration as well as various environmental stresses. Using the parameters calculated from work within our labs and the data available in published literature the model of the Ku mediated D-NHEJ pathway and the PARP-1 mediated B-NHEJ pathway was found at rest to produce very similar results to the live MRC5 cells with over half the breaks being resolved in less than 2 hours (Figure 6A) and the majority of remaining foci being resolved within 8 hours.

Results

Conclusion