Histone H2AX phosphorylation in normal human cells irradiated with focused ultrasoft X rays: evidence for chromatin movement during repair.

DNA repair within the cell nucleus is a dynamic process involving a close interaction between repair proteins and chromatin structure. Recent studies have indicated a quantitative relationship between DNA double-strand break induction and histone H2AX phosphorylation. The dynamics of this process within individual cell nuclei is unknown. To address this, we have used a novel focused ultrasoft X-ray microprobe that is capable of inducing localized DNA damage within a subnuclear area of intact cells with a 2.5-microm-diameter beam spot. The present investigation was undertaken to explore the influence of focused irradiation of individual nuclei with 1.49 keV characteristic aluminum K-shell (AlK) X rays on H2AX phosphorylation in normal human cells. Immunofluorescence analyses revealed that significant diffusion of the initial spots of clustered foci of phosphorylated H2AX occurred in a time-dependent fashion after exposure to AlK X rays. Irradiation under cooled conditions resulted in a reduction in the size of spots of clustered foci of phosphorylated H2AX as well as of individual phosphorylated H2AX foci. These findings strongly suggest that diffusion of the chromatin microenvironment occurs during the repair of DNA damage. We also found that AlK ultrasoft X rays (71 foci per gray) were 2.2-fold more effective at the initial formation of phosphorylated H2AX foci than with conventional X rays (32 foci per gray), and that the time required to eliminate 50% of the initial number of foci was 3.4-fold longer in AlK-irradiated cells than that in cells exposed to conventional X rays. For conventional X rays, we also report significant accumulation of larger-sized foci at longer times after irradiation.