Intragenomic variability and extended sequence patterns in the mutational signature of ultraviolet light.

Mutational signatures can reveal properties of underlying mutational processes and are important when assessing signals of selection in cancer. Here, we describe the sequence characteristics of mutations induced by ultraviolet (UV) light, a major mutagen in several human cancers, in terms of extended (longer than trinucleotide) patterns as well as variability of the signature across chromatin states. Promoter regions display a distinct UV signature with reduced TCG > TTG transitions, and genome-wide mapping of UVB-induced DNA photoproducts (pyrimidine dimers) showed that this may be explained by decreased damage formation at hypomethylated promoter CpG sites. Further, an extended signature model encompassing additional information from longer contextual patterns improves modeling of UV mutations, which may enhance discrimination between drivers and passenger events. Our study presents a refined picture of the UV signature and underscores that the characteristics of a single mutational process may vary across the genome.

DNA polymerase η contributes to genome-wide lagging strand synthesis.

DNA polymerase η (pol η) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol η also has other cellular roles. Here, we present evidence that pol η competes with DNA polymerases α and δ for the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol η, which contains a PCNA-Interacting Protein motif is required for pol η to function in lagging strand synthesis. Finally, we provide evidence that a pol η dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol η and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.

Elevated pyrimidine dimer formation at distinct genomic bases underlies promoter mutation hotspots in UV-exposed cancers.

Sequencing of whole cancer genomes has revealed an abundance of recurrent mutations in gene-regulatory promoter regions, in particular in melanoma where strong mutation hotspots are observed adjacent to ETS-family transcription factor (TF) binding sites. While sometimes interpreted as functional driver events, these mutations are commonly believed to be due to locally inhibited DNA repair. Here, we first show that low-dose UV light induces mutations preferably at a known ETS promoter hotspot in cultured cells even in the absence of global or transcription-coupled nucleotide excision repair (NER). Further, by genome-wide mapping of cyclobutane pyrimidine dimers (CPDs) shortly after UV exposure and thus before DNA repair, we find that ETS-related mutation hotspots exhibit strong increases in CPD formation efficacy in a manner consistent with tumor mutation data at the single-base level. Analysis of a large whole genome cohort illustrates the widespread contribution of this effect to recurrent mutations in melanoma. While inhibited NER underlies a general increase in somatic mutation burden in regulatory elements including ETS sites, our data supports that elevated DNA damage formation at specific genomic bases is at the core of the prominent promoter mutation hotspots seen in skin cancers, thus explaining a key phenomenon in whole-genome cancer analyses.

Base-resolution UV footprinting by sequencing reveals distinctive damage signatures for DNA-binding proteins.

Decades ago, it was shown that proteins binding to DNA can quantitatively alter the formation of DNA damage by UV light. This established the principle of UV footprinting for non-intrusive study of protein-DNA contacts in living cells, albeit at limited scale and precision. Here, we perform deep base-resolution quantification of the principal UV damage lesion, the cyclobutane pyrimidine dimer (CPD), at select human promoter regions using targeted CPD sequencing. Several transcription factors exhibited distinctive and repeatable damage signatures indicative of site occupancy, involving strong (up to 17-fold) position-specific elevations and reductions in CPD formation frequency relative to naked DNA. Positive damage modulation at some ETS transcription factor binding sites coincided at base level with melanoma somatic mutation hotspots. Our work provides proof of concept for the study of protein-DNA interactions at individual loci using light and sequencing, and reveals widespread and potent modulation of UV damage in regulatory regions.

Somatic mutation distribution across tumour cohorts provides a signal for positive selection in cancer.

Cancer gene discovery is reliant on distinguishing driver mutations from a multitude of passenger mutations in tumour genomes. While driver genes may be revealed based on excess mutation recurrence or clustering, there is a need for orthogonal principles. Here, we take advantage of the fact that non-cancer genes, containing only passenger mutations under neutral selection, exhibit a likelihood of mutagenesis in a given tumour determined by the tumour's mutational signature and burden. This relationship can be disrupted by positive selection, leading to a difference in the distribution of mutated cases across a cohort for driver and passenger genes. We apply this principle to detect cancer drivers independently of recurrence in large pan-cancer cohorts, and show that our method (SEISMIC) performs comparably to traditional approaches and can provide resistance to known confounding mutational phenomena. Being based on a different principle, the approach provides a much-needed complement to existing methods for detecting signals of selection.