UV-induced DNA lesions are important contributors to mutagenesis and cancer, but it is not fully understood how the chromosomal landscape influences UV lesion formation and repair. Genome-wide profiling of repair activity in UV irradiated cells has revealed significant variations in repair kinetics across the genome, not only among large chromatin domains, but also at individual transcription factor binding sites. Here we report that there is also a striking but predictable variation in initial UV damage levels across a eukaryotic genome. We used a new high-throughput sequencing method, known as CPD-seq, to precisely map UV-induced cyclobutane pyrimidine dimers (CPDs) at single-nucleotide resolution throughout the yeast genome. This analysis revealed that individual nucleosomes significantly alter CPD formation, protecting nucleosomal DNA with an inward rotational setting, even though such DNA is, on average, more intrinsically prone to form CPD lesions. CPD formation is also inhibited by DNAbound transcription factors, in effect shielding important DNA elements from UV damage. Analysis of CPD repair revealed that initial differences in CPD damage formation often persist, even at later repair time points. Furthermore, our high-resolution data demonstrate, to our knowledge for the first time, that CPD repair is significantly less efficient at translational positions near the dyad of strongly positioned nucleosomes in the yeast genome. These findings define the global roles of nucleosomes and transcription factors in both UV damage formation and repair, and have important implications for our understanding of UV-induced mutagenesis in human cancers.DNA repair | DNA damage | nucleosome | chromatin | transcription factor U ltraviolet (UV) light causes extensive damage to DNA by inducing the formation of cyclobutane pyrimidine dimers (CPDs) and, to a lesser extent, 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs). If unrepaired, these DNA lesions block normal DNA replication and are major contributors to mutagenesis in skin cancers (1). CPDs and 6-4PPs are primarily repaired in cells by the nucleotide excision repair (NER) pathway (1). CPD lesions in actively transcribed strands (TS) of DNA are repaired rapidly by the transcription coupled-NER (TC-NER) branch of this repair pathway, which is triggered by RNA polymerase II stalling at UV damage (2, 3). In contrast, CPD lesions in the remainder of the genome are repaired by the global genome NER (GG-NER) subpathway. Differences in repair rates between transcribed and nontranscribed DNA, and between accessible and inaccessible chromatin domains, have been invoked to explain the mutational heterogeneity in many cancer genomes (4-6). To gain new insight into the mutational processes that lead to human cancer, however, a more detailed understanding is needed of the complex interplay of UV damage formation and repair across the genome.Our understanding of how NER operates in different sequence and chromatin contexts has been aided by two recent genome-wide surveys of NER ac...

Recurrent mutations are frequently associated with transcription factor (TF) binding sites (TFBS) in melanoma, but the mechanism driving mutagenesis at TFBS is unclear. Here, we use a method called CPD-seq to map the distribution of UV-induced cyclobutane pyrimidine dimers (CPDs) across the human genome at single nucleotide resolution. Our results indicate that CPD lesions are elevated at active TFBS, an effect that is primarily due to E26 transformation-specific (ETS) TFs. We show that ETS TFs induce a unique signature of CPD hotspots that are highly correlated with recurrent mutations in melanomas, despite high repair activity at these sites. ETS1 protein renders its DNA binding targets extremely susceptible to UV damage in vitro, due to binding-induced perturbations in the DNA structure that favor CPD formation. These findings define a mechanism responsible for recurrent mutations in melanoma and reveal that DNA binding by ETS TFs is inherently mutagenic in UV-exposed cells.

SUMMARY Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. Here, we investigate the dynamic protein-DNA interactions during the damage recognition step using single-molecule fluorescence microscopy. Quantum dot-labeled Rad4-Rad23 (yeast XPC-RAD23B ortholog) forms nonmotile complexes or conducts a one-dimensional search via either random diffusion or constrained motion. Atomic force microcopy analysis of Rad4 with the β-hairpin domain 3 (BHD3) deleted reveals that this motif is non-essential for damage-specific binding and DNA bending. Furthermore, we find that deletion of seven residues in the tip of β-hairpin in BHD3 increases Rad4-Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance.

DNA base damage is an important contributor to genome instability, but how the formation and repair of these lesions is affected by the genomic landscape and contributes to mutagenesis is unknown. Here, we describe genome-wide maps of DNA base damage, repair, and mutagenesis at single nucleotide resolution in yeast treated with the alkylating agent methyl methanesulfonate (MMS). Analysis of these maps revealed that base excision repair (BER) of alkylation damage is significantly modulated by chromatin, with faster repair in nucleosome-depleted regions, and slower repair and higher mutation density within strongly positioned nucleosomes. Both the translational and rotational settings of lesions within nucleosomes significantly influence BER efficiency; moreover, this effect is asymmetric relative to the nucleosome dyad axis and is regulated by histone modifications. Our data also indicate that MMS-induced mutations at adenine nucleotides are significantly enriched on the nontranscribed strand (NTS) of yeast genes, particularly in BER-deficient strains, due to higher damage formation on the NTS and transcription-coupled repair of the transcribed strand (TS). These findings reveal the influence of chromatin on repair and mutagenesis of base lesions on a genome-wide scale and suggest a novel mechanism for transcription-associated mutation asymmetry, which is frequently observed in human cancers.

UV radiation induces photolesions that distort the DNA double helix and, if not repaired, can cause severe biological consequences, including mutagenesis or cell death. In eukaryotes, both the formation and repair of UV damage occur in the context of chromatin, in which genomic DNA is packaged with histones into nucleosomes and higher-order chromatin structures. Here, we review how chromatin impacts the formation of UV photoproducts in eukaryotic cells. We describe the initial discovery that nucleosomes and other DNA-binding proteins induce characteristic ‘photofootprints’ during the formation of UV photoproducts. We also describe recent progress in genome-wide methods for mapping UV damage, which echoes early biochemical studies, and highlights the role of nucleosomes and transcription factors in UV damage formation and repair at unprecedented resolution. Finally, we discuss our current understanding of how the distribution and repair of UV-induced DNA damage influence mutagenesis in human skin cancers.