4.7 Article

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

Journal

Publisher

MDPI
DOI: 10.3390/ijms222010957

Keywords

DNA double-strand breaks; DNA-damage-response; chromatin; chromatin dynamics; hypotonic stress (HypoS); homologous recombination (HR); nonhomologous end-joining (NHEJ); alternative end-joining (alt-EJ); single-strand annealing (SSA); ionizing radiation (IR)

Funding

  1. German Federal Ministry of Education and Research [BMBF02NUK037B, BMBF-02NUK043B, BMBF-02NUK054B]
  2. German Research Foundation [DFGGRK1739]
  3. German Federal Ministry for Economic Affairs [BMWi-50WB1836]

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The decompaction of chromatin induced by hypotonic stress affects the processing and repair of DNA double-strand breaks (DSBs), leading to increased sensitivity of cells to ionizing radiation-induced damage and compromising genomic stability. This is achieved through suppression of homologous recombination (HR) and enhancement of error-prone processing by alternative end-joining (alt-EJ) and single-strand annealing (SSA).
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, gamma-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 G(2)-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.

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