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Noncoding mutation hotspots have been identified in melanoma and many of them occur at the binding sites of E26 transformation-specific (ETS) proteins; however, their formation mechanism and functional impacts are not fully understood. Here, we used UV (Ultraviolet) damage sequencing data and analyzed cyclobutane pyrimidine dimer (CPD) formation, DNA repair, and CPD deamination in human cells at single-nucleotide resolution. Our data show prominent CPD hotspots immediately after UV irradiation at ETS binding sites, particularly at sites with a conserved TTCCGG motif, which correlate with mutation hotspots identified in cutaneous melanoma. Additionally, CPDs are repaired slower at ETS binding sites than in flanking DNA. Cytosine deamination in CPDs to uracil is suggested as an important step for UV mutagenesis. However, we found that CPD deamination is significantly suppressed at ETS binding sites, particularly for the CPD hotspot on the 5′ side of the ETS motif, arguing against a role for CPD deamination in promoting ETS-associated UV mutations. Finally, we analyzed a subset of frequently mutated promoters, including the ribosomal protein genesRPL13AandRPS20, and found that mutations in the ETS motif can significantly reduce the promoter activity. Thus, our data identify high UV damage and low repair, but not CPD deamination, as the main mechanism for ETS-associated mutations in melanoma and uncover important roles of often-overlooked mutation hotspots in perturbing gene transcription. 

DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method, N -methylpurine-sequencing (NMP-seq), and characterized alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data show that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs ARS binding factor 1 (Abf1) and rDNA enhancer binding protein 1 (Reb1), but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro, causing slow repair both within the core motif and its adjacent DNA. Repair of ultraviolet (UV) damage by nucleotide excision repair (NER) was also inhibited by TF binding. Interestingly, TF binding inhibits a larger DNA region for NER relative to BER. The observed effects are caused by the TF–DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.