UV exposure causes not only cytosine-to-thymine (C > T) substitutions in dipyrimidine sequences but also many non-canonical mutation classes (e.g. A > T, T > C, and AC > TT). While C > T substitutions are thought to arise from mutagenic bypass of cyclobutane pyrimidine dimers (CPDs), the photoproduct(s) that cause non-canonical mutation classes, which are responsible for key driver mutations in melanoma, are unclear. Here, we use lesion-specific photolyases and whole genome sequencing of yeast irradiated with predominately UVB light to dissect the origins of different classes of UV mutations. These data reveal that CPDs are responsible for ∼60% of all UV mutations and cause not only C > T mutations but also a subset of non-canonical mutation classes, particularly in CT sequence contexts. Our data also indicate that UV-induced pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are responsible for slightly less than half of all UV-induced mutations in yeast and are the primary cause of T > C substitutions in TT sequences and C > A substitutions. Finally, ∼5% of UV mutations are resistant to both CPD and 6-4PP photolyases, and these are comprised of A > T and AC > TT substitutions in purine-containing dinucleotides. Collectively, these findings define the photoproducts responsible for different UV mutation classes across a eukaryotic genome and indicate that many A > T and AC > TT substitutions arise from atypical UV photoproducts.

Persistence of common ultraviolet (UV)–induced lesions, like cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4-PPs), typically results in C>T substitutions at dipyrimidines: a mutation pattern that composes the single-base substitution (SBS) signature 7 in cancer. Oncogenic melanoma mutations rarely involve SBS7-like substitutions. We recently identified noncanonical UV-induced mutations in yeast that appear to originate from atypical AC and TA photoproducts. While an AC photoproduct could account for formation of BRAF V600K, other melanoma drivers like BRAF V600E and NRAS Q61K involve other mutation types, suggesting possible existence of additional atypical photoproducts. Here, we couple temperature-induced telomeric end resection in yeast with serial UV irradiation and whole-genome sequencing to show UV light induces an extended array of noncanonical mutations in single-stranded DNA (ssDNA). This includes A T >A M , G T >G V , A C >A A , A T> T T, and T A >T T substitutions that are resistant to photo-reversion, indicating that they likely originate from atypical photoproducts. UV-induced mutation spectra in yeast lacking Rad30 indicated that Pol η plays substantial roles in the bypass of CPDs and 6-4-PPs regardless of telomere proximity. Unexpectedly, expression of a mutant DNA pol ε (pol2 M644G) reduced both canonical and noncanonical UV-induced mutations specifically within subtelomeric regions of the genome. This suggests a preferential role for pol ε in the resynthesis of uncapped telomeres, with the M644G mutation conferring accurate lesion bypass capabilities to the replicative polymerase. ssDNA-specific UV lesions provide additional damage-mediated mechanisms for the production of oncogenic mutations in melanoma, such as the BRAF V600E mutation that involves a G T >G A substitution.

Mutation hotspots in melanoma frequently occur at DNA binding sites of E26 transformation-specific (ETS)-family transcription factors, as ETS factors stimulate the formation of UV-induced cyclobutane pyrimidine dimers (CPDs) while suppressing repair at ETS-bound DNA sites. To elucidate the molecular mechanism by which ETS factors bind to damaged DNA sites and inhibit repair, we investigated the binding of members from the three major classes of the ETS superfamily (Ets1, ELF1, and PU.1) to cognate DNA containing a cis-syn TpT CPD. These site-specific CPDs modulated ETS recognition and repair by a model repair enzyme in a position-dependent manner. Specifically, a deaminated CPD located in a damage hotspot in the ETS binding motif consistently stimulated binding and inhibited T4 PDG (a CPD repair enzyme) by all three paralogs. Co-crystal structures of PU.1 reveal that CPDs and mismatches are recognized within the framework of canonical ETS/DNA complexes. Molecular dynamics simulations in explicit solvent show that CPD introduces compensatory structural dynamics to both the free and ETS-bound states that strongly modify the underlying thermodynamics of recognition. The results offer a molecular basis for how ETS factors induce mutation hotspots in skin cancers and other UV-exposed tissues by binding to CPD-containing sites and inhibiting their repair.

UV light induces cyclobutane pyrimidine dimers (CPDs) and other mutagenic lesions in cellular DNA. Cytosine-containing CPDs can subsequently undergo rapid deamination to uracil, a process that has been linked to UV mutagenesis. However, the impact of genomic context and chromatin architecture on CPD deamination rates in cells remains poorly understood. Here, we develop a method known as dCPD-seq to map deaminated CPDs (dCPDs) across the genome of repair-deficient yeast cells at single-nucleotide resolution. Our dCPD-seq data reveal that sequence context significantly modulates CPD deamination rates in UV-irradiated yeast cells, with CPDs in TCG contexts showing particularly rapid deamination rates. Our analysis indicates that rapid CPD deamination can explain why UV-induced mutations are specifically enriched at TCG sequences, both in UV-irradiated yeast cells and in human skin cancers. CPD deamination is suppressed near the transcription start and end sites of yeast genes, which may in part by mediated by DNA-bound transcription factors. Finally, we show that the wrapping of DNA in nucleosomes modulates CPD deamination in yeast cells. Our data indicate that CPD deamination is elevated at minor-in rotational positions where the DNA minor groove faces the histone octamer, likely owing to increased solvent accessibility of the C4 position of the cytosine base. Moreover, we also observe strand-specific enrichment of CPD deamination at rotational positions where the DNA backbone faces out toward the solvent. Taken together, these findings reveal how DNA sequence context and chromatin architecture modulates CPD deamination rates across a eukaryotic genome.

Abstract Genomic studies revealed elevated DNA damage and mutation rates at transcription factor (TF) binding sites in UV-linked cancers. While TFs can promote UV-induced mutagenesis by altering both damage formation and repair, these mechanisms have not been systematically characterized across TFs at high resolution. Using genome-wide UV damage maps from skin fibroblasts, we develop a scalable statistical framework to analyze TF-mediated mutagenic mechanisms across hundreds of TFs. We identify numerous previously unreported TFs that significantly enhance or suppress UV damage formation within their binding sites. A systematic survey of TF-DNA complexes reveals that damage modulation often coincides with TF-induced DNA distortions that either protect against or promote photodimer formation. Additionally, we analyze repair efficiency in TF binding sites at high resolution, identifying TFs likely to compete with repair. Comparisons with skin cancer mutations distinguish mutation enrichment driven by increased damageability versus attenuated repair, revealing the highly contextual nature of TF-mediated mutagenesis.

Benzo[a]pyrene is a carcinogen in tobacco smoke that, when metabolized to benzo[a]pyrene diol epoxide (BPDE), induces mutagenic DNA lesions that promote the development of lung cancer. In lung cells, BPDE damages DNA packaged in nucleosomes, but the impact of nucleosomes on BPDE adduct formation is unclear. Here, we analyze genome-wide maps of BPDE adduct formation and repair in human cells. Our analysis indicates that BPDE adduct formation is suppressed in nucleosomes and enriched in adjacent linker DNA. Within nucleosomes, BPDE adduct formation is specifically elevated at minor-out rotational settings, where the minor groove of the DNA faces outward from the histone octamer. Structural analysis indicates that the solvent accessibility of the reactive exocyclic N2 amino group in guanine bases is elevated at minor-out rotational settings, potentially accounting for elevated BPDE damage at these locations. These damage patterns coincide with and can explain elevated somatic mutation rates in lung cancers at linker DNA and minor-out rotational settings in nucleosomes. While BPDE damage formation in nucleosomes strongly correlates with mutation patterns in lung cancers, the repair of these adducts does not. Analysis of damage patterns at CCCTC-binding factor and SP1 transcription factor binding sites indicates that BPDE damage formation is also suppressed by these DNA-bound proteins, and this damage modulation correlates with mutation patterns at these binding sites in lung cancers. These data indicate that altered BPDE adduct formation in chromatin can explain the distinct patterns of somatic mutations in lung cancers.

Exposure to the ultraviolet (UV) spectrum of sunlight poses a threat to terrestrial species. Nearly all species possess the nucleotide excision repair (NER) machinery, which can repair the helix-distorting DNA lesions induced by UV light. However, many species also have photolyase enzymes, which use near-UV and visible wavelengths of sunlight to directly reverse major classes of UV photoproducts. In eukaryotic cells, both of these repair pathways must efficiently locate and repair UV photoproducts present in chromatin. While genome-wide damage mapping methods have been used to extensively characterize how chromatin and ongoing transcription impact NER, much less is known about how photolyase enzymes navigate these obstacles to repair UV damage. Here, we highlight a recent article from our laboratory that used genome-wide sequencing methods to characterize how yeast photolyase repairs UV damage, both in NER-proficient and -deficient cells, and prevents UV-induced mutations.

Cyclobutane pyrimidine dimers (CPDs) are formed in DNA following exposure to ultraviolet (UV) light and are mutagenic unless repaired by nucleotide excision repair (NER). It is known that CPD repair rates vary in different genome regions owing to transcription-coupled NER and differences in chromatin accessibility; however, the impact of regional chromatin organization on CPD formation remains unclear. Furthermore, nucleosomes are known to modulate UV damage and repair activity, but how these damage and repair patterns are affected by the overarching chromatin domains in which these nucleosomes are located is not understood. Here, we generated a new CPD damage map inDrosophilaS2 cells using CPD-seq and analyzed it alongside existing excision repair-sequencing (XR-seq) data to compare CPD damage formation and repair rates across five previously established chromatin types inDrosophila. This analysis revealed that repair activity varies substantially across different chromatin types, whereas CPD formation is relatively unaffected. Moreover, we observe distinct patterns of repair activity in nucleosomes located in different chromatin types, which we show is owing to domain-specific differences in nucleosome repeat length (NRL). These findings indicate that NRL is altered in different chromatin types inDrosophilaand that changes in NRL modulate the repair of UV lesions.

Genomic studies have revealed elevated damage and mutation rates in active transcription factor (TF) binding sites in UV-linked cancers. Previous investigations into the relationship between TF activity and UV DNA damage have primarily focused on select TFs or been done in aggregate across large cohorts of TFs at kilobase resolution. While collectively, there is evidence that TFs contribute to UV-induced mutagenesis by both enhancing initial damage formation and attenuating repair, there has yet to be a comprehensive characterization of these mechanisms on a per-TF basis. Using genome-wide maps of UV damage from human skin fibroblasts, we developed a scalable statistical framework to analyze TF-mediated mutagenic mechanisms across hundreds of TFs. We identify numerous previously unreported TFs that significantly enhance and / or inhibit damage formation in their binding sites. A systematic survey of TF-DNA complexes further revealed that positions of UV damage modulation coincide with TF-induced structural distortions that either protect or predispose DNA to photodimer formation. Additionally, we analyzed repair efficiency in TF binding sites with unprecedented resolution, identifying specific TFs and binding site positions likely to compete with repair factors. By comparing these results with skin cancer mutations, we distinguish mutation peaks driven by increased damage susceptibility versus attenuated repair, illustrating that TF-mediated mutagenesis is highly contextual and dependent on the TF, binding site position, and sequence context of the damaged locus. Our approach provides a robust statistical framework for elucidating mechanisms of mutagenic TF-binding and offers novel insights into the complex interplay between protein interactions, DNA damage, and repair.

Deciphering transcriptional networks requires methods to accurately map binding sites of sequence-specific transcription factors (ssTFs) across the genome. Here, we show that ssTF binding induces distinct patterns of UV-induced cyclobutane pyrimidine dimers (CPDs), and that these CPD 'fingerprints' can be exploited by machine learning methods to identify ssTF binding sites (TFBS). As a proof of principle, we analyzed CPD-seq data from yeast cells using the Random Forest algorithm to identify 75 TFBS bound by the Hap2/Hap3/Hap5 ssTF complex, including ∼25 new sites missed by previous chromatin immunoprecipitation (ChIP)-based experiments. Parallel analysis of the Gcr1 ssTF using a neural network trained on CPD-seq data including only 6 known binding sites identified 63 Gcr1 TFBS across the genome. Our analysis indicates that the newly identified TFBS are associated with many genes that function in expected categories (e.g. mitochondrial respiration or glycolysis), and whose mRNA levels are down-regulated in ssTF mutants. Similar analysis of CPD-capture-sequencing data from human cells identified new sites bound by the homologous Nuclear Factor-Y complex. These findings indicate that distinct cellular patterns of UV damage occurring at different classes of TFBS can be recognized by machine learning methods to map these regulatory elements with improved accuracy and single-nucleotide resolution.

Replication timing during S-phase impacts mutation rates in yeast and human cancers; however, the exact mechanism involved remains unclear. Here, we analyze the impact of replication timing on UV mutagenesis in Saccharomyces cerevisiae. Our analysis indicates that UV mutations are enriched in early-replicating regions of the genome in wild-type cells, but in cells deficient in global genomic-nucleotide excision repair (GG-NER), mutations are enriched in late-replicating regions. Analysis of UV damage maps revealed that cyclobutane pyrimidine dimers are enriched in late-replicating regions, but this enrichment is almost entirely due to repetitive ribosomal DNA. Complex mutations typically associated with TLS activity are also elevated in late-replicating regions in GG-NER deficient cells. We propose that UV mutagenesis is higher in early-replicating regions in repair-competent cells because there is less time to repair the lesion prior to undergoing replication. However, in the absence of GG-NER, increased TLS activity promotes UV mutagenesis in late-replicating regions.

Ultraviolet (UV) light is the most pervasive environmental mutagen and the primary cause of skin cancer. Genome sequencing of melanomas and other skin cancers has revealed that the vast majority of somatic mutations in these tumors are cytosine-to-thymine (C>T) substitutions in dipyrimidine sequences, which, together with tandem CC>TT substitutions, comprise the canonical UV mutation "signature". These mutation classes are caused by DNA damage directly induced by UV absorption, namely cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone photoproducts (6-4PP), which form between neighboring pyrimidine bases. However, many of the key driver mutations in melanoma do not fit this mutation signature, but instead are caused by T>A, T>C, C>A, or AC>TT substitutions, frequently occurring in non-dipyrimidine sequence contexts. This article describes recent studies indicating that UV light causes a more diverse spectrum of mutations than previously appreciated, including many of the mutation classes observed in melanoma driver mutations. Potential mechanisms for these diverse mutation signatures are discussed, including UV-induced pyrimidine-purine photoproducts and indirect DNA damage induced by UVA light. Finally, the article reviews recent findings indicating that human DNA polymerase eta normally suppresses these non-canonical UV mutation classes, which can potentially explain why canonical C>T substitutions predominate in human skin cancers.

DNA repair pathways play a critical role in genome stability, but in eukaryotic cells, they must operate to repair DNA lesions in the compact and tangled environment of chromatin. Previous studies have shown that the packaging of DNA into nucleosomes, which form the basic building block of chromatin, has a profound impact on DNA repair. In this review, we discuss the principles and mechanisms governing DNA repair in chromatin. We focus on the role of histone post-translational modifications (PTMs) in repair, as well as the molecular mechanisms by which histone mutants affect cellular sensitivity to DNA damage agents and repair activity in chromatin. Importantly, these mechanisms are thought to significantly impact somatic mutation rates in human cancers and potentially contribute to carcinogenesis and other human diseases. For example, a number of the histone mutants studied primarily in yeast have been identified as candidate oncohistone mutations in different cancers. This review highlights these connections and discusses the potential importance of DNA repair in chromatin to human health.

Nucleotide excision repair (NER) removes helix-distorting DNA lesions and is therefore critical for genome stability. During NER, DNA is unwound on either side of the lesion and excised, but the rules governing incision site selection, particularly in eukaryotic cells, are unclear. Excision repair-sequencing (XR-seq) sequences excised NER fragments, but analysis has been limited because the lesion location is unknown. Here, we exploit accelerated cytosine deamination rates in UV-induced CPD (cyclobutane pyrimidine dimer) lesions to precisely map their locations at C to T mismatches in XR-seq reads, revealing general and species-specific patterns of incision site selection during NER. Our data indicate that the 5' incision site occurs preferentially in HYV (i.e. not G; C/T; not T) sequence motifs, a pattern that can be explained by sequence preferences of the XPF-ERCC1 endonuclease. In contrast, the 3' incision site does not show strong sequence preferences, once truncated reads arising from mispriming events are excluded. Instead, the 3' incision is partially determined by the 5' incision site distance, indicating that the two incision events are coupled. Finally, our data reveal unique and coupled NER incision patterns at nucleosome boundaries. These findings reveal key principles governing NER incision site selection in eukaryotic cells.

Transcription coupled-nucleotide excision repair (TC-NER) removes DNA lesions that block RNA polymerase II (Pol II) transcription. A key step in TC-NER is the recruitment of the TFIIH complex, which initiates DNA unwinding and damage verification; however, the mechanism by which TFIIH is recruited during TC-NER, particularly in yeast, remains unclear. Here, we show that the C-terminal domain (CTD) of elongation factor-1 (Elf1) plays a critical role in TC-NER in yeast by binding TFIIH. Analysis of genome-wide repair of UV-induced cyclobutane pyrimidine dimers (CPDs) using CPD-seq indicates that the Elf1 CTD in yeast is required for efficient TC-NER. We show that the Elf1 CTD binds to the pleckstrin homology (PH) domain of the p62 subunit of TFIIH in vitro, and identify a putative TFIIH-interaction region (TIR) in the Elf1 CTD that is important for PH binding and TC-NER. The Elf1 TIR shows functional, structural, and sequence similarities to a conserved TIR in the mammalian UV sensitivity syndrome A (UVSSA) protein, which recruits TFIIH during TC-NER in mammalian cells. These findings suggest that the Elf1 CTD acts as a functional counterpart to mammalian UVSSA in TC-NER by recruiting TFIIH in response to Pol II stalling at DNA lesions.