Investigations into the spatial distribution of ?H2AX around a DNA double-strand break and the analysis of double-strand break mobility

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation by the protein kinase ATM. H2AX is unevenly distributed throughout chromatin and is rapidly phosphorylated to form ?H2AX up to 2 megabases either side of DSBs. Studies in yeast systems have shown that while ?H2A can spread in cis surrounding the break site, it can also spread in trans onto unbroken chromosomes located in close spatial proximity. Although the majority of data in the current literature presents the well characterised in cis spread of ?H2AX, there are strong indications that it can also occur in trans in mammalian systems; analogous to the findings shown in yeast. This thesis lays out the steps taken to develop a novel system to address the spatial distribution of ?H2AX around a nascent DSB. Since the first published live imaging experiments of the dynamics of chromatin by in vivo single particle tracking there has been extensive investigation into the regulation and biological function of movement of damaged DNA. In yeast, a relative consensus exists that DSB induction increases the movement of a DSB. In contrast to yeast however, data published of DSB movement in higher eukaryotes has been controversial, caused by conflicting results. Here, I developed a cell-based system, and utilised timelapse live cell imaging to show that a chromosomal locus containing a single endonuclease-induced DSB shows confined movement in comparison to an undamaged locus. Furthermore, this confined movement of a damaged locus is compounded by treatment with an ATM kinase inhibitor but not a DNA-PKcs kinase inhibitor, suggesting that the kinase activity of ATM and not the kinase activity of DNA-PKcs plays a significant role in the dynamics of DSBs.