Mechanisms of activation of H2AX phosphorylation.

The cellular response to DNA damage is aimed at protecting organisms from harmful effects of unrepaired or mis-repaired damage, such as mutagenesis. One component of DNA damage signaling is the phosphorylation of H2AX, a histone variant that is randomly distributed in the genome, by activated kinases like ATM, ATR and DNA-PKcs. The mechanisms of H2AX phosphorylation and its exact function are not fully understood. After DNA damage, H2AX is phosphorylated rapidly and in a dose-dependent manner, in discrete areas of chromatin, that can be observed as sub-nuclear foci by immunofluorescent microscopy. gammaH2AX foci are suggested to mark sites of DNA double strand breaks (DSBs) and to participate in their repair. However, H2AX also gets phosphorylated after exposure to agents that do not directly induce DSB, such as ultraviolet light (UV). In this dissertation, the potential mechanisms for the induction of gammaH2AX after UV were investigated. It was found that after UV exposure, H2AX phosphorylation occurred independently of replication and appeared to be triggered by repair processing of UV-induced lesions. Thus, H2AX phosphorylation was markedly increased when repair intermediates were accumulated in normal human fibroblasts by blockage of the rejoining step of nucleotide excision repair. The ATR kinase was found to play a major role in mediating the phosphorylation. Interestingly, repair deficient cells also exhibited H2AX phosphorylation at later times after UV. It was hypothesized that this late induction may be triggered by transcription inhibition by UV-induced lesions. Indeed, it was found that blockage specifically of transcription elongation led to H2AX phosphorylation and also phosphorylation of the tumor suppressor p53 at serine15. In conclusion, this thesis demonstrates that formation of gammaH2AX is not limited to double-strand breaks, but can also occur as a result of DNA damage processing and blockage of transcription elongation. Thus, it extends our current understanding of how phosphorylation of the histone variant H2AX is triggered. H2AX phosphorylation may play a role in modulating chromatin structure to both enhance DNA repair and to facilitate restoration of DNA topology following repair. This chromatin function may be the missing link that connects H2AX phosphorylation to the maintenance of genomic integrity.PhDBiological SciencesHealth and Environmental SciencesMolecular biologyOncologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126707/2/3276175.pd