Investigating the effects of repair of DNA single-strand breaks on chromatin structure

Single-strand breaks (SSBs) are one of the most common types of lesion arising within cells; formed by attack of genotoxic agents on the DNA, as well as enzymatically during normal cellular processes. Although the single-strand break repair (SSBR) pathway is relatively well characterised, and many components have been extensively studied in vitro, little is known of how this pathway operates in vivo when DNA is complexed with histone proteins to form chromatin. This compaction of the DNA into nucleosomal structures has the potential to inhibit repair, by sterically blocking access of repair factors to sites of DNA damage. Whilst previous studies have shown that repair of DNA double-strand breaks and UV-induced lesions are associated with alterations in chromatin structure, through covalent modification of histone proteins and nucleosome remodeling, few similar observations have been made concerning SSBR. Here, I have produced and employed mammalian cell lines stably expressing fluorescently-tagged histone proteins to analyse the dynamics of chromatin occurring upon DNA damage. Localised damage was introduced using micro-irradiation with a UV-A laser, and the histone proteins at the site of damage visualized in real-time using confocal microscopy. Through this method, I have identified a rearrangement of chromatin structure in the vicinity of DNA strand breaks in mammalian cells, resulting in a mobilization of histone proteins at the site of damage. Furthermore, I have shown that this alteration is partially dependent on the activities of both the SSBR factor poly(ADP-ribose) polymerase 1 (PARP-1), and the phosphoinositide 3-kinase-like kinase (PIKK) Ataxia telangiectasia mutated (ATM). I have examined a potential requirement for ATM in SSBR, and found no evidence of this, suggesting that the effects of PARP-1 and ATM on histone mobilization are reflective of the independent contributions of repair of single- and double-strand breaks respectively.