The team used correlative fluorescence and energy-filtering microscopy to watch what happens to chromatin after DSBs occur. The technique measures the amount of specific elements, in this case nitrogen and phosphorous, at each point in the sample. By looking at the ratio of the two elements, the researchers can distinguish between proteins and nucleic acids, and thus can watch how the different nuclear components behave in living cells.
The chromatin adjacent to DSBs remained in the same nuclear location even several hours after DNA breakage. However, higher-resolution imaging showed that the chromatin fibers became somewhat decondensed. Surprisingly, the chromatin opening and recruitment of repair factors to DSBs was energy dependent and did not proceed in ATP-depleted cells, though it is not yet clear which complex or complexes require the energy during the process.
Immediately after the introduction of DSBs, phosphorylated ATM, a key protein in the DNA damage repair signaling pathway, and phosphorylated histone H2AX, which has been shown to be required for assembling repair proteins, appeared at the breaks. Chromatin decondensation occurred normally in cells lacking either ATM or H2AX, but the repair complex was unstable in H2AX-null cells and dissipated prematurely.
The resolution of the new data is too low to determine what is happening within the DNA immediately adjacent to the break; there might be significant freedom of movement in the nanometer range. But the data do show that there is not sufficient movement in mammalian cells to allow multiple DSBs to congregate during repair, which may explain why translocations between nonneighboring chromosomal locations are relatively rare.