Disruption of Chromatin Dynamics by Hypotonic Stress Suppresses HR and Shifts DSB Processing to Error-Prone SSA.

Lisa Marie Krieger,George Iliakis,Emil Mladenov,Marilen Demond,Martin Stuschke,Aashish Soni

doi:10.3390/ijms222010957

Abstract

The processing of DNA double-strand breaks (DSBs) depends on the dynamic characteristics of chromatin. To investigate how abrupt changes in chromatin compaction alter these dynamics and affect DSB processing and repair, we exposed irradiated cells to hypotonic stress (HypoS). Densitometric and chromosome-length analyses show that HypoS transiently decompacts chromatin without inducing histone modifications known from regulated local chromatin decondensation, or changes in Micrococcal Nuclease (MNase) sensitivity. HypoS leaves undisturbed initial stages of DNA-damage-response (DDR), such as radiation-induced ATM activation and H2AX-phosphorylation. However, detection of ATM-pS1981, γ-H2AX and 53BP1 foci is reduced in a protein, cell cycle phase and cell line dependent manner; likely secondary to chromatin decompaction that disrupts the focal organization of DDR proteins. While HypoS only exerts small effects on classical nonhomologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ), it markedly suppresses homologous recombination (HR) without affecting DNA end-resection at DSBs, and clearly enhances single-strand annealing (SSA). These shifts in pathway engagement are accompanied by decreases in HR-dependent chromatid-break repair in the G2-phase, and by increases in alt-EJ and SSA-dependent chromosomal translocations. Consequently, HypoS sensitizes cells to ionizing radiation (IR)-induced killing. We conclude that HypoS-induced global chromatin decompaction compromises regulated chromatin dynamics and genomic stability by suppressing DSB-processing by HR, and allowing error-prone processing by alt-EJ and SSA.

Highlights

Each cell has to deal with tens of thousands of DNA lesions every day [1] that can cause mutations endangering genomic stability
Transfer of actively growing retinal epithelial cells (RPE) cells to hypotonic medium stresses cells by causing water influx that increases their volume by about 5% within 5 min (Figure 1a)
Since analysis by Western blot (Figure 3a) showed normal activation of ATM in irradiated cells exposed to hypotonic stress (HypoS), we considered the possibility that the absence of visible pATM foci in Figure 3d,e is secondary to HypoS-induced chromatin decondensation, i.e., ATM is present at double-strand breaks (DSBs) at normal levels but becomes dispersed on decondensed chromatin, failing to appear as discernible foci

Summary

Introduction

Each cell has to deal with tens of thousands of DNA lesions every day [1] that can cause mutations endangering genomic stability. One consequential form of DNA damage is the DNA double-strand break (DSB). Cells have evolved different repair pathways to process DSBs, all with the ability to structurally restore the DNA molecule, but each operating with different fidelity and bearing different risks to the genome [4]. Cells have evolved highly sophisticated and complex signaling mechanisms to detect DSBs and promote their repair, collectively known as the DNAdamage-response (DDR) [2,5]. When components of this signaling and repair network are defective, hypersensitivity to DNA damaging agents and diverse disease phenotypes develop [2]

Methods

Results

Conclusion