For the first time, DNA double-strand breaks (DSBs) were directly visualized in functionally and structurally different chromatin domains of human cells. The results show that genetically inactive condensed chromatin is much less susceptible to DSB induction by gamma-rays than expressed, decondensed domains. Higher sensitivity of open chromatin for DNA damage was accompanied by more efficient DSB repair. These findings follow from comparing DSB induction and repair in two 11 Mbp-long chromatin regions, one with clusters of highly expressed genes and the other, gene-poor, containing mainly genes having only low transcriptional activity. The same conclusions result from experiments with whole chromosome territories, differing in gene density and consequently in chromatin condensation. It follows from our further results that this lower sensitivity of DNA to the damage by ionizing radiation in heterochromatin is not caused by the simple chromatin condensation but very probably by the presence of a higher amount of proteins compared to genetically active and decondensed chromatin. In addition, our results show that some agents potentially used for cell killing in cancer therapy (TSA, hypotonic and hypertonic) influence cell survival of irradiated cells via changes in chromatin structure and efficiency of DSB repair in different ways.

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We show that double strand breaks (DSBs) induced in chromatin of low as well as high density by exposure of human cells to gamma-rays are repaired in low-density chromatin. Extensive chromatin decondensation manifested in the vicinity of DSBs by decreased intensity of chromatin labelling, increased H4K5 acetylation, and decreased H3K9 dimethylation was observed already 15 min after irradiation. Only slight movement of sporadic DSB loci for short distances was noticed in living cells associated with chromatin decondensation around DSBs. This frequently resulted in their protrusion into the low-density chromatin domains. In these regions, the clustering (contact or fusion) of DSB foci was seen in vivo, and in situ after cell fixation. The majority of these clustered foci were repaired within 240 min, but some of them persisted in the nucleus for several days after irradiation, indicating damage that is not easily repaired. We propose that the repair of DSB in clustered foci might lead to misjoining of ends and, consequently, to exchange aberrations. On the other hand, the foci that persist for several days without being repaired could lead instead to cell death.