γ-H2AX Foci Persistence at Chromosome Break Suggests Slow and Faithful Repair Phases Restoring Chromosome Integrity.

Michelle Ricoul,Tamizh Selvan Gnana Sekaran,Patricia Brochard,Laure Sabatier,Cecile Herate

doi:10.3390/cancers11091397

Abstract

Many toxic agents can cause DNA double strand breaks (DSBs), which are in most cases quickly repaired by the cellular machinery. Using ionising radiation, we explored the kinetics of DNA lesion signaling and structural chromosome aberration formation at the intra- and inter-chromosomal level. Using a novel approach, the classic Premature Chromosome Condensation (PCC) was combined with γ-H2AX immunofluorescence staining in order to unravel the kinetics of DNA damage signalisation and chromosome repair. We identified an early mechanism of DNA DSB joining that occurs within the first three hours post-irradiation, when dicentric chromosomes and chromosome exchanges are formed. The slower and significant decrease of ”deleted chromosomes” and 1 acentric telomere fragments observed until 24 h post-irradiation, leads to the conclusion that a second and error-free repair mechanism occurs. In parallel, we revealed remaining signalling of γ-H2AX foci at the site of chromosome fusion long after the chromosome rearrangement formation. Moreover there is important signalling of foci on the site of telomere and sub-telomere sequences suggesting either a different function of γ-H2AX signalling in these regions or an extreme sensibility of the telomere sequences to DNA damage that remains unrepaired 24 h post-irradiation. In conclusion, chromosome repair happens in two steps, including a last and hardly detectable one because of restoration of the chromosome integrity.

Highlights

Scoring of chromosome rearrangements is largely applied to determine and quantify genotoxic effects of environmental components, pharmaceutical drugs, or ionizing radiation
Due to the timing of reparation, the study of the double strand breaks (DSBs) repair mechanism kinetics needs an immediate visualisation of condensed chromosomes after break induction while irradiated peripheral blood lymphocytes (PBL) are mainly in the G0 phase [26]
Abbreviations used in this figure and others are explained in the section “Abbreviations”. γ-H2AX foci are considered as early DNA DSB signalling both on hamster and human Premature Chromosome Condensation (PCC) (Figure 1A)

Summary

Introduction

Scoring of chromosome rearrangements is largely applied to determine and quantify genotoxic effects of environmental components, pharmaceutical drugs, or ionizing radiation. Clastogenic effects of such agents will lead to double strand breaks (DSBs) and later chromosome rearrangements during the DNA repair phase. The cells are employing different mechanisms in order to repair the DSB depending on the cell cycle phase, and this can lead in some cases to chromosome aberrations (CA). We focus on the exchange-type aberrations, i.e., dicentrics and translocations. The dicentric chromosomes result from the fusion of two broken chromosomes with one centromere, and contain two centromeres. It is usually associated with acentric fragments without centromere. The translocated chromosomes result from the association of two chromosomes restoring an apparent structure integrity (one centromere and telomeres at each extremity).

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Discussion

Conclusion