Formation of Dynamic γ-H2AX Domains along Broken DNA Strands Is Distinctly Regulated by ATM and MDC1 and Dependent upon H2AX Densities in Chromatin

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate γ-H2AX. Here, we demonstrate that γ-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which γ-H2AX forms does not spread as DSBs persist; rather, γ-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate γ-H2AX, only ATM promotes γ-H2AX formation to maximal distance and maintains γ-H2AX densities. MDC1 is essential for γ-H2AX formation at high densities near DSBs, but not for generation of γ-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of γ-H2AX formed. Our data suggest that H2AX fuels a γ-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX.