Genomic Sites Ultrasensitive to Ultraviolet Radiation

If the genome contains outlier sequences extraordinarily sensitive to environmental agents, these would be sentinels for monitoring personal carcinogen exposure and might drive direct changes in cell physiology rather than acting through rare mutations. New methods, adductSeq and freqSeq, provided statistical resolution to quantify rare lesions at single-base resolution across the genome. Primary human melanocytes, but not fibroblasts, carried spontaneous apurinic sites and TG sequence lesions more frequently than UV-induced cyclobutane pyrimidine dimers (CPDs). UV exposure revealed hyperhotspots acquiring CPDs up to 170 fold more frequently than the genomic average; these sites were more prevalent in melanocytes. Hyperhotspots were disproportionately located near genes, particularly for RNA-binding proteins, with the most-recurrent hyperhotspots at a fixed position within two motifs: one occurring at ETS1 transcription factor binding sites, known to be UV targets, and at sites of mTOR/TOP-tract translation regulation; the second occurring at A2-15TTCTY, which developed "dark CPDs" after UV exposure, repaired CPDs slowly, and had accumulated CPDs prior to the experiment. Motif locations active as hyperhotspots differed between cell types. Melanocyte CPD hyperhotspots aligned precisely with recurrent UV signature mutations in individual gene promoters of melanomas and with known cancer drivers. At sunburn levels of UV exposure, every cell would have a hyperhotspot CPD in each of the ~20 targeted cell pathways, making hyperhotspots act as epigenetic marks. Purpose: These experiments searched for genomic sites in human primary fibroblasts and melanocytes that are extraordinarily sensitive to DNA damage, primarily cyclobutane pyrimidine dimers (CPDs) induced by UVC or UVB radiation. They separately detected abasic sites and other spontaneous DNA damage when present. In most experiments, pre-existing abasic sites were first blocked by nicking with an apurinic endonuclease; in all experiments, shear breaks were blocked using phosphatase. Then DNA was incised with a DNA repair enzyme recognizing CPDs. CPDs were reversed enzymatically and linkers were ligated to the 5' phosphate at the nick. Libraries were constructed and sequenced by paired-end Illumina sequencing to identify linker-ligated sites. Statistical analysis compared +UV data to -UV data and pyrimidine-pyrimidine sites (putative CPD sites) to PuPu sites (controls for copy number and starting cell number), to calculate CPD frequencies at individual bases. Please note that each processed data file was generated from multiple samples (as indicated in the corresponding sample description field). Several baseline samples were generated to establish the baseline of the technique itself and thus no processed data was generated. Although the baseline samples are not used directly in any of the final conclusions, they are included in the records as discussed heavily in the associated manuscript.