Reinardy HC, Dharamshi J, Jha AN, Henry TB
DNA repair is initiated by transcription of genes in response to specific types of damage. Breaks in DNA strands (single and double) are repaired predominantly through non-homologous end-joining (NHEJ) or homologous recombination (HR), but progression of repair and changes in expression profiles of genes involved are unknown. DNA damage was induced in zebrafish larvae by brief exposure (10min) to hydrogen peroxide (H2O2; 100mM), and induction of DNA strand breaks was assessed by single-cell gel electrophoresis (comet) assay over 24h. H2O2 was selected because it is eliminated rapidly after induction of DNA damage. DNA damage [mean ± standard error of the mean (SEM), % tail DNA] increased significantly immediately after 10-min H2O2 exposure (35.4±3.8; control 17.2±2.0), but damage did not differ from control levels 24h after exposure (9.2±0.4; control 9.9±0.9). At 0-, 1-, 3-, 6-, 12- and 24-h post-exposure, quantitative reverse transcriptase–PCR was conducted to assess expression of selected genes involved in DNA repair including xrcc5, xrcc6 (NHEJ), rad51 (HR) and gadd45a (DNA damage detection). Expression (maximum fold-change ± SEM, triplicate samples of 40 larvae) of each gene increased rapidly (within 6h) after exposure to 100mM of H2O2: 1.8±0.2, rad51; 1.7±0.2, xrcc5 and 1.5±0.1, xrcc6. Acute exposure (200mM of H2O2) caused 10% larval mortality within 2h, upregulated gadd45a (5.0±0.8), but did not change expression of rad51, xrcc5 or xrcc6. Expression profiles (critical exponential model) were similar among genes but differed relative to time and among independent experiments. Results indicate that repair mechanisms are initiated rapidly after DNA damage, that gene expression profiles vary according to potency of H2O2 exposure and that examination of the time course of gene expression changes is necessary to understand the complete gene response over time.
Reinardy HC, Dharamshi J, Jha AN, Henry TB. 2013. Changes in expression profiles of genes associated with DNA repair following induction of DNA damage in larval zebrafish Danio rerio. Mutagenesis 28:601-608.