Mechanistic Modelling of Slow and Fast NHEJ DNA Repair Pathways Following Radiation for G0/G1 Normal Tissue Cells

Abstract

Mechanistic in silico models can provide insight into biological mechanisms and highlight uncertainties for experimental investigation. Radiation-induced double-strand breaks (DSBs) are known to be toxic lesions if not repaired correctly. Non-homologous end joining (NHEJ) is the major DSB-repair pathway available throughout the cell cycle and, recently, has been hypothesised to consist of a fast and slow component in G0/G1. The slow component has been shown to be resection-dependent, requiring the nuclease Artemis to function. However, the pathway is not yet fully understood. This study compares two hypothesised models, simulating the action of individual repair proteins on DSB ends in a step-by-step manner, enabling the modelling of both wild-type and protein-deficient cell systems. Performance is benchmarked against experimental data from 21 cell lines and 18 radiation qualities. A model where resection-dependent and independent pathways are entirely separated can only reproduce experimental repair kinetics with additional restraints on end motion and protein recruitment. However, a model where the pathways are entwined was found to effectively fit without needing additional mechanisms. It has been shown that DaMaRiS is a useful tool when analysing the connections between resection-dependent and independent NHEJ repair pathways and robustly matches with experimental results from several sources.

Immediate-Early, Early, and Late Responses to DNA Double Stranded Breaks

“…Once bound, KU70/80 not only facilitates synapsis but also protects DNA ends from resection, thereby promoting cNHEJ. Emerging data supports a division of cNHEJ into two distinct biphasic pathways, termed fast-kinetic and slow-kinetic cNHEJ (Figures 1B, 2B,E, and Jakob et al, 2011;Biehs et al, 2017;Chang et al, 2017;Löbrich and Jeggo, 2017;Shibata et al, 2018;Frit et al, 2019;Setiaputra and Durocher, 2019;Shibata and Jeggo, 2020a;Shibata and Jeggo, 2020b;Qi et al, 2021). The fastkinetic cNHEJ is also termed 53BP1-, Artemis-, or resectionindependent cNHEJ, with Artemis clearly function downstream of ATM (Riballo et al, 2004;Woodbine et al, 2011).…”

“…In addition to the complex competition and recruitment interactions between KU70/80 and PARP, KU70/80 and MRN also share what has been termed 'entwined' loading, meaning they are not loaded in defined sequential order or competitively, but rather with more complex dynamics that include multiple points of crosstalk (see below and Rupnik et al, 2008;Shibata et al, 2018;Ingram et al, 2019). In addition, the common model where KU70/80 solely promotes NHEJ by recruiting DNA-PK, and MRN promotes HR by recruiting ATM, is clearly an oversimplification, as both complexes can be loaded to the same DSB (Britton et al, 2013;Ingram et al, 2019;Qi et al, 2021).…”

“…Whereas the core cNHEJ proteins are required by both fast-and slow-kinetic cNHEJ, 53BP1, RIF1, and Shieldin are antiresection factors and CST-Polα primase balances resection with de novo DNA synthesis, likely improving fidelity (Mirman et al, 2018). However, fast-and slow-kinetic cNHEJ differ in their ability to repair simple versus complex DSBs, have different recruitment pathways, and are used to different extents throughout the cell cycle (Setiaputra and Durocher, 2019;Shibata and Jeggo, 2020a;Shibata and Jeggo, 2020b;Qi et al, 2021). Intriguingly, emerging data suggests that slow-kinetic cNHEJ can avoid mutagenic deletions by using RNA molecules as homology templates for retrieving sequence information that can be lost during resection (Storici et al, 2007;Chakraborty et al, 2016;Meers et al, 2016;Mazina et al, 2017).…”

An updated view into the cell cycle kinetics of human T lymphocytes and the impact of irradiation

“…These findings suggest that the cells did not undergo cell cycle arrest after exposure to 1 and 2 Gy of X-rays in the G0 phase, as this would have resulted in a decreased proportion of dividing cells due to arrest at the cell cycle checkpoints 34 . This indicates that most of the DNA damage induced by irradiation was repaired before continuing their cell cycle progression, which is not unexpected as it is known that the main repair pathway active in the G0 phase, namely the non-homologous end joining (NHEJ) pathway, ensures repair of DNA damage within 24 h 35 , 36 . It is important to note that in these experiments, the cells were exposed to moderate doses of X-rays and that higher doses (> 7.5 Gy) have been reported to inhibit the proliferative response of lymphocytes, as shown by multiple studies 37 , 38 .…”

Modeling of DNA Damage Repair and Cell Response in Relation to p53 System Exposed to Ionizing Radiation

“…The process of DNA repair can be simulated mechanically according to the information of lesions. Stochastic methods [ 21 , 22 ] and deterministic methods [ 1 , 4 , 23 , 24 ] are used in the simulation of DNA repair. Several models consider the resection-independent and resection-dependent NHEJ [ 22 , 25 ].…”