Figure 5

hMYH and hSIRT6 are recruited to oxidatively damaged sites located within transcriptionally active chromatin (TA-KR), but not within inactive chromatin (tetR-KR) in human U2OS TRE cells. a Scheme of tetR- and TA-tagged KR expression in the U2OS TRE cell [41]. To induce ROS-mediated damage at a specific locus in the human genome, we fused KR to the tetracycline repressor (tetR) to induce ROS damage in a 90-kb TRE array (total of 200 repeats) in U2OS cells. We also fused KR to the transcription activator tetR + VP16 (TA) to examine damage response at active chromatin. GFP-hMYH or GFP-hSIRT6 was co-transfected into the cells to analyze the recruitment of these proteins to the oxidative DNA damage sites. b, c GFP-MYH and GFP-SIRT6 are not enriched at sites with TA-mCherry in undamaged cells. d, e Damage response of GFP-MYH and GFP-SIRT6 to the site of TA-KR after light activation. f, g No recruitment of GFP-MYH and GFP-SIRT6 to the site of tetR-KR after light activation. Analyses of about 50 cells in each KR activated group indicated that over 90% of cells showed the colocalization of GFP-MYH foci or GFP-SIRT6 foci with TA-KR, in contract, none of the MYH or SIRT6 expressing cell showed foci at sites of tetR-KR.