Abstract
Reliable and efficient detection of DNA damage constitutes a vital capability of human cells to maintain genome stability. Following DNA damage, the histone variant H2AX becomes rapidly phosphorylated by the DNA damage response kinases DNA-PKcs and ATM. H2AX phosphorylation plays a central role in signal amplification leading to chromatin remodeling and DNA repair initiation. The contribution of DNA-PKcs and ATM to H2AX phosphorylation is however puzzling. Although ATM is required, DNA-PKcs can substitute for it. Here we analyze the interplay between DNA-PKcs and ATM with a computational model derived by an iterative workflow: switching between experimental design, experiment and model analysis, we generated an extensive set of time-resolved data and identified a conclusive dynamic signaling model out of several alternatives. Our work shows that DNA-PKcs and ATM enforce a biphasic H2AX phosphorylation. DNA-PKcs can be associated to the initial, and ATM to the succeeding phosphorylation phase of H2AX resulting into a signal persistence detection function for reliable damage sensing. Further, our model predictions emphasize that DNA-PKcs inhibition significantly delays H2AX phosphorylation and associated DNA repair initiation.
Highlights
Cells are constantly affected by DNA damage, resulting from ionizing g-irradiation (IR), genotoxic or replication stress and reactive oxygen species
We report an iterative workflow combining experimental work, computational modeling and experimental design methodologies to shed light on the interplay of two phosphoinositide 3-kinase like kinase (PIKK) family members (DNAPKcs and ataxia telangiectasia mutated (ATM)) to the rapid histone H2AX phosphorylation in the context of DNA damage sensing upon g-irradiation
By performing optimized dynamic stimulation experiments, we generated an extensive set of time-resolved data to identify a computational model for analyzing DNA-PKcs-P, ATM-P and gH2AX dynamics
Summary
Cells are constantly affected by DNA damage, resulting from ionizing g-irradiation (IR), genotoxic or replication stress and reactive oxygen species. DNA damage, including single and double strand breaks (DSB), base modification, deletions or point mutations, seriously affects genome stability and cell integrity if not properly detected and repaired by the DNA damage response (DDR).[1]. Serine 139 (gH2AX) to generate foci at the DSB site.[3] The assembly of chromatin remodeling complexes at the DSB site greatly depends on gH2AX and enables the accessibility of the damaged DNA to repair proteins.[4]. Family; ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). ATR phosphorylates H2AX upon replicative stress,[5] whereas ATM and DNA-PKcs are responsible for this phosphorylation upon DNA DSB, which are induced by IR.[6]. ATM and DNA-PKcs have been studied on a qualitative basis focusing on their impact of repair pathway choice for rebuilding damaged DNA either via rapid (classical) non-homologous end joining cNHEJ and/or slow homologous recombination repair (HR) pathway.[7,8] As for the pathway choice, the interplay between
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