Fig. 5
Binding of ETS1 protein promotes UV damage formation in vitro. a DNA sequences of RPL13A and SDHD promoter fragments, corresponding to chromosome coordinates chr19:49990710-49990681 and chr11:111957515-111957553, respectively. Putative ETS motifs are shown in gray background and highlighted in bold. Recurrent mutated sites in melanomas are underlined. b, c Gel shift assays showing binding of purified ETS1 protein to radiolabeled RPL13A (b) and SDHD (c) promoter fragments, respectively. d A representative sequencing gel (15%) showing CPD formation in naked RPL13A (sample 1, with UV irradiation) and ETS1-bound RPL13A DNA (samples 2–5, UV irradiation). The binding products shown in part (b) were irradiated with 1KJ m−2 of UV-C light and CPD lesions were converted to single strand breaks by T4 endonuclease V digestion. The resulting DNA breaks were separated on a 15% denaturing sequencing gel to analyze damage abundance at different locations. A negative control (naked DNA without UV irradiation) was also digested with T4 endoV to show the background level of DNA cleavage in the absence of UV-induced DNA lesions. The first lane on the left shows a 10-nt DNA ladder. e Same as in part (d), except the SDHD promoter fragment was analyzed on a 12% gel. Asterisk indicates gel running artifact caused by bromophenol blue in the gel loading buffer. Both RPL13A and SDHD CPD formation experiments were conducted at least 3 times independently with consistent results
