Transcription factor binding enhances UV DNA Damage at specific loci within their binding sites

Transcription factors (TFs) bind to specific sequences throughout the genome to regulate associated genes. Though thought to be well conserved, sites where TFs are bound (TFBSs) have high frequencies of sequence variation. This suggests that TF binding is inherently mutagenic. There is evidence for two models for how this can occur: 1) enhanced damage; 2) occlusion of repair. Both lead to an accumulation of damage and eventual mutation. The focus of this study is on whether, and to what extent, TFs enhance DNA damage using UV as a source of damage. We studied two different TFs, Ets1 and GR. Six different variations of the Ets1 consensus sequence and nine different variations of the GR consensus sequence were synthesized to test for enhancement of damage of specific di-pyrimidines in specific positions under different sequence contexts. Purified recombinant Ets1 and GR were bound to their respective sequences. These sequences were irradiated with UVC light and incubated with UVE1, an endonuclease that nicks 5’ to DNA damage. Nicked sequences were separated on a urea denaturing gel and DNA damage was quantified. Our results showed that the frequency of DNA damage is altered upon TF binding. In the Ets1 binding sites, TC (-4, -3) had a ~16-fold change in damage while other positions had a ~2-fold change of enhancement. When the identity of this di-pyrimidine was changed, enhanced damage was still observed indicating that the position of di-pyrimidine is what leads to DNA damage enhancement. At GR binding sites, damage enhancement was observed but was markedly lower. The enhancement of damage was influenced by position and sequence context. Damage patterns of both TFs were compared to predicted structures, but no significant correlations were found with any of the measured parameters. This work shows that more than one TF can enhance damage of DNA, though some have more modest effects.