γH2AX foci on apparently intact mitotic chromosomes: Not signatures of misrejoining events but signals of unresolved DNA damage

The presence of H2AX foci on apparently intact mitotic chromosomes is controversial because they challenge the assumed relationship between H2AX foci and DNA double-strand breaks (DSBs). In this work, we show that after irradiation during interphase, a variety of H2AX foci are scored in mitotic cells. Surprisingly, approximately 80% of the H2AX foci spread over apparently undamaged chromatin at Terminal or Interstitial positions and they can display variable sizes, thus being classified as Small, Medium and Big foci. Chromosome and chromatid breaks that reach mitosis are spotted with Big (60%) and Medium (30%) Terminal H2AX foci, but very rarely are they signaled with Small H2AX foci. To evaluate if Interstitial H2AX foci might be signatures of misrejoining, an mFISH analysis was performed on the same slides. The results show that Interstitial H2AX foci lying on apparently intact chromatin do not mark sites of misrejoining, and that misrejoined events were never signaled by a H2AX foci during mitosis. Finally, when analyzing the presence of other DNA-damage response (DDR) factors we found that all H2AX fociregardless their coincidence with a visible breakalways colocalized with MRE11, but not with 53BP1. This pattern suggests that these H2AX foci may be hallmarks of both microscopically visible and invisible DNA damage, in which an active, although incomplete or halted DDR is taking place.