The major mechanism of melanoma mutations is based on deamination of cytosine in pyrimidine dimers as determined by circle damage sequencing

Abstract

Sunlight-associated melanomas carry a unique C-to-T mutation signature. UVB radiation induces cyclobutane pyrimidine dimers (CPDs) as the major form of DNA damage, but the mechanism of how CPDs cause mutations is unclear. To map CPDs at single-base resolution genome wide, we developed the circle damage sequencing (circle-damage-seq) method. In human cells, CPDs form preferentially in a tetranucleotide sequence context (5'-Py-T<>Py-T/A), but this alone does not explain the tumor mutation patterns. To test whether mutations arise at CPDs by cytosine deamination, we specifically mapped UVB-induced cytosine-deaminated CPDs. Transcription start sites (TSSs) were protected from CPDs and deaminated CPDs, but both lesions were enriched immediately upstream of the TSS, suggesting a mutation-promoting role of bound transcription factors. Most importantly, the genomic dinucleotide and trinucleotide sequence specificity of deaminated CPDs matched the prominent mutation signature of melanomas. Our data identify the cytosine-deaminated CPD as the leading premutagenic lesion responsible for mutations in melanomas.

Fig. 1. Outline of circle-damage-seq.

(A) DNA containing damaged bases (red) is sonicated, circularized, and then treated with an exonuclease cocktail. (B) DNA lesions (arrows) are processed into DNA double-strand breaks (DSB) by base excision repair enzymes and S1 nuclease. Sequencing adaptors are ligated to the breaks. (C) DNA library preparation for DNA damage–specific sequence enrichment. Divergent paired-end reads with single-base gaps indicate the damaged base positions. (D) Structure of a CPD at a TC sequence. (E) Method for mapping of UVB-induced CPDs at single-nucleotide resolution. (F) Divergent paired reads of CPD mapping by circle-damage-seq. Purple and lavender segments represent reads mapped to the plus and minus strands, respectively. Green arrows indicate single-base gaps matching the 5′ base of a pyrimidine dimer. ATP, adenosine triphosphate; DNase, deoxyribonuclease.

Fig. 2. Sequence context of CPD formation in human fibroblasts irradiated with UVB.

(A) Distribution plot of trinucleotide sequences (PyPyN3′) undergoing CPD formation in UVB-irradiated human cells (green). NT, nontreated control (red). Some background is seen in the NT samples at non-dipyrimidine sequences (the four trinucleotides to the right). (B) Distribution plot of trinucleotide sequences (5′NPyPy) undergoing CPD formation in UVB-irradiated human cells (green). Data in (A) and (B) are normalized for the trinucleotide frequencies of the human genome.

Fig. 3. CPD mapping by circle-damage-seq shows the distribution of CPDs at the sequence and gene level.

(A) Nucleotide composition of mapped CPD positions. Position 11 and 12 represent UVB-damaged dipyrimidine sequences undergoing CPD formation. At each base position, the height of each letter represents the relative frequency of that nucleic acid base. (B) G+C sequence enrichment along human genes (hg19). (C) Heatmap and metagene profiles of CPD distribution along all genes of the hg19 human genome. CPD coverage is sorted from high (top, blue) to low (bottom, red). The CPD signals were mapped and binned in 50-bp windows from 1.5 kb upstream of the transcription start site (TSS) and then normalized relative to gene length over the gene bodies to the transcription end site (TES) and 1.5 kb downstream of the TES. (D) Heatmap and profile of CPD distribution around the TSS and 1.5 kb of flanking sequence of all hg19 genes. (E) Heatmap and profile of CPD distribution around the TES and 1.5 kb of flanking sequence of all hg19 genes. (F) Example of CPD sequence read distribution along several genes on chr2. There is a reduced frequency of CPDs near the TSS (blue arrows).

Fig. 4. Mapping of deaminated CPDs at the sequence and gene level.

(A) Outline of the method used for mapping of deaminated CPDs. The red “=” symbol indicates a CPD. (B) Divergent paired reads of deaminated CPDs obtained by circle-damage-seq. Purple and lavender segments represent reads mapped to the plus and minus strands, respectively. Green arrows indicate single-base gaps at cytosine bases matching the deaminated base of a pyrimidine dimer. The trinucleotide context of the deaminated cytosine is indicated. (C) Heatmaps and metagene profiles of deaminated CPDs in UVB-irradiated human fibroblasts. Total signal is sorted from high to low (top to bottom). Metagene profiles are shown for untreated cells (NT) and at 0, 24, and 48 hours following UVB irradiation. (D) Browser views of total CPDs (top) and cytosine-deaminated CPDs (bottom) along the DKK3 gene on chromosome 11 in human fibroblasts. Note the reduced levels of deaminated CPDs at the TSS containing a CGI (red bar).

Fig. 5. Levels of total CPDs and deaminated CPDs at TSSs and TESs.

(A) Human genes with G+C-rich promoters (CGIs) show low frequencies of total CPDs and deaminated CPDs near TSS (red arrows) and increased levels just upstream of the TSS. (B) Human genes without CGI promoters do not show CPD depletion near the TSS but still show reduced levels of deaminated CPDs near the TSS albeit at a lesser extent than at CGIs (see scales). (C) Heatmap and metagene profile of total CPDs and deaminated CPDs near the TES ± 1.5 kb in human UVB-irradiated fibroblasts. The data in this figure were prepared using 100 million aligned, divergent read pairs with single-base gaps.

Fig. 6. Sequence specificity of deaminated CPDs in the UVB-irradiated human genome and melanoma mutations.

(A) Outline of CPD deamination and potential mutagenic consequences. (B) Sequence context of the occurrence of deaminated CPDs at 48 hours following UVB irradiation. Position 11 is the deaminated cytosine. (C) Trinucleotide sequence specificity of total CPD formation, formation of deaminated CPDs, and the prominent mutational signature 7 (SBS7) in melanoma. For total CPDs, we show the sequences in the context of 5′N-dipyrimidine (NPyPy; blue line plot, ranked according to levels; CPDs at NPyPy are underlined). For deaminated CPDs and mutations, the NPyN (n = 32) trinucleotide context is shown (green and red bars, respectively). (D) Trinucleotide sequence specificity of total CPD formation, formation of deaminated CPDs, and the mutation signature SBS7 in melanoma genomes. We show the sequences in the context 5′dipyrimidine-N (PyPyN) (n = 16). Data in (B) to (D) are not normalized for total genomic trinucleotide frequencies (see also fig. S6). (E) Heatmap showing the cosine similarity scores for total CPDs (NPyPy, n = 32), deaminated CPDs, and the prominent mutational signature 7 (SBS7/SBS7a/SBS7b) in melanoma along with the 30 COSMIC v2 mutational signatures. We only used the C-to-T and T-to-C mutation windows for comparisons with the COSMIC trinucleotide signatures. Dark blue colors indicate high similarity (see also fig. S7).