Chromatin Oxidation Disrupts DNA Replication and Engages Base Excision Repair
Excessive reactive oxygen species can result in significant macromolecular damage. This includes oxidation of chromatin, the key organizing structure of the genome. While many groups have studied the DNA damage response following general oxidant treatment (i.e. H₂O₂ or KBrO₃) this oxidation is not specific to chromatin and the type of DNA damage produced varies, obscuring how cells process specific oxidative lesions and their effects on cell health.
Our group utilizes H2B-FAP, a chemoptogenetic tool to specifically oxidize chromatin. When cells expressing H2B-FAP are treated with a photosensitizer dye and 660nm light, singlet oxygen is produced in chromatin and we confirmed this results in the common lesion, 8-oxo-guanine.
In both cancerous and non-cancerous cells, chromatin oxidation results in dose-dependent reductions in cell growth. We are currently investigating the mechanistic causes of this, and find find significant single-strand DNA accumulation following oxidation, leading to RPA recruitment and activation of ATR signaling. Interestingly, we see immediate activation of ATM signaling as well, but no detectable double-strand breaks by neutral comet assays, until 6 hours post oxidation. We hypothesize that inefficient base excision repair of 8oxoG is resulting in replication fork breakage and are developing approaches to study this in real-time in live cells.