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

The presence of γH2AX foci on apparently intact mitotic chromosomes is controversial because they challenge the assumed relationship between γH2AX foci and DNA double-strand breaks (DSBs). In this work, we show that after irradiation during interphase, a variety of γH2AX foci are scored in mitotic cells. Surprisingly, approximately 80% of the γH2AX 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 γH2AX foci, but very rarely are they signaled with Small γH2AX foci. To evaluate if Interstitial γH2AX foci might be signatures of misrejoining, an mFISH analysis was performed on the same slides. The results show that Interstitial γH2AX foci lying on apparently intact chromatin do not mark sites of misrejoining, and that misrejoined events were never signaled by a γH2AX foci during mitosis. Finally, when analyzing the presence of other DNA-damage response (DDR) factors we found that all γH2AX foci—regardless their coincidence with a visible break—always colocalized with MRE11, but not with 53BP1. This pattern suggests that these γH2AX foci may be hallmarks of both microscopically visible and invisible DNA damage, in which an active, although incomplete or halted DDR is taking place.

γH2AX 焦点的存在在看似完整的有丝分裂染色体上存在争议，因为它们挑战了 γH2AX 焦点与 DNA 双链断裂（DSBs）之间假设的关系。在本研究中，我们发现，在间期照射后，有丝分裂细胞中评分了多种 γH2AX 焦点。令人惊讶的是，大约 80% 的 γH2AX 焦点分布在看似未受损的染色质上，位于端部或间部位置，并且它们可以呈现不同的尺寸，从而被归类为小型、中型和大型焦点。达到有丝分裂的染色体和染色单体断裂在大型（60%）和中型（30%）端部 γH2AX 焦点中被检测到，但它们很少用小型 γH2AX 焦点来标记。为了评估间部 γH2AX 焦点是否可能是错误连接的标志，我们对同一张切片进行了 mFISH 分析。结果显示，位于看似完整染色质上的间部 γH2AX 焦点并不标记错误连接的位点，并且错误连接事件在有丝分裂过程中从未由 γH2AX 焦点来标记。最后，在分析其他 DNA 损伤反应（DDR）因子的存在时，我们发现所有 γH2AX 焦点——无论它们是否与可见的断裂相吻合——总是与 MRE11 共定位，而不与 53BP1 共定位。这一模式表明，这些 γH2AX 焦点可能是显微可见和不可见 DNA 损伤的标志，其中正在进行着一种活跃的、尽管不完整或已停止的 DDR。