Analysis of cytosine deamination events in excision repair sequencing reads reveals mechanisms of incision site selection in NER.

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.

Genome-wide maps of rare and atypical UV photoproducts reveal distinct patterns of damage formation and mutagenesis in yeast chromatin.

Ultraviolet (UV) light induces different classes of mutagenic photoproducts in DNA, namely cyclobutane pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and atypical thymine-adenine photoproducts (TA-PPs). CPD formation is modulated by nucleosomes and transcription factors (TFs), which has important ramifications for Ultraviolet (UV) mutagenesis. How chromatin affects the formation of 6-4PPs and TA-PPs is unclear. Here, we use UV damage endonuclease-sequencing (UVDE-seq) to map these UV photoproducts across the yeast genome. Our results indicate that nucleosomes, the fundamental building block of chromatin, have opposing effects on photoproduct formation. Nucleosomes induce CPDs and 6-4PPs at outward rotational settings in nucleosomal DNA but suppress TA-PPs at these settings. Our data also indicate that DNA binding by different classes of yeast TFs causes lesion-specific hotspots of 6-4PPs or TA-PPs. For example, DNA binding by the TF Rap1 generally suppresses CPD and 6-4PP formation but induces a TA-PP hotspot. Finally, we show that 6-4PP formation is strongly induced at the binding sites of TATA-binding protein (TBP), which is correlated with higher mutation rates in UV-exposed yeast. These results indicate that the formation of 6-4PPs and TA-PPs is modulated by chromatin differently than CPDs and that this may have important implications for UV mutagenesis.

CTCF binding modulates UV damage formation to promote mutation hot spots in melanoma.

Somatic mutations in DNA-binding sites for CCCTC-binding factor (CTCF) are significantly elevated in many cancers. Prior analysis has suggested that elevated mutation rates at CTCF-binding sites in skin cancers are a consequence of the CTCF-cohesin complex inhibiting repair of UV damage. Here, we show that CTCF binding modulates the formation of UV damage to induce mutation hot spots. Analysis of genome-wide CPD-seq data in UV-irradiated human cells indicates that formation of UV-induced cyclobutane pyrimidine dimers (CPDs) is primarily suppressed by CTCF binding but elevated at specific locations within the CTCF motif. Locations of CPD hot spots in the CTCF-binding motif coincide with mutation hot spots in melanoma. A similar pattern of damage formation is observed at CTCF-binding sites in vitro, indicating that UV damage modulation is a direct consequence of CTCF binding. We show that CTCF interacts with binding sites containing UV damage and inhibits repair by a model repair enzyme in vitro. Structural analysis and molecular dynamic simulations reveal the molecular mechanism for how CTCF binding modulates CPD formation.

Set2 histone methyltransferase regulates transcription coupled-nucleotide excision repair in yeast.

Helix-distorting DNA lesions, including ultraviolet (UV) light-induced damage, are repaired by the global genomic-nucleotide excision repair (GG-NER) and transcription coupled-nucleotide excision repair (TC-NER) pathways. Previous studies have shown that histone post-translational modifications (PTMs) such as histone acetylation and methylation can promote GG-NER in chromatin. Whether histone PTMs also regulate the repair of DNA lesions by the TC-NER pathway in transcribed DNA is unknown. Here, we report that histone H3 K36 methylation (H3K36me) by the Set2 histone methyltransferase in yeast regulates TC-NER. Mutations in Set2 or H3K36 result in UV sensitivity that is epistatic with Rad26, the primary TC-NER factor in yeast, and cause a defect in the repair of UV damage across the yeast genome. We further show that mutations in Set2 or H3K36 in a GG-NER deficient strain (i.e., rad16Δ) partially rescue its UV sensitivity. Our data indicate that deletion of SET2 rescues UV sensitivity in a GG-NER deficient strain by activating cryptic antisense transcription, so that the non-transcribed strand (NTS) of yeast genes is repaired by TC-NER. These findings indicate that Set2 methylation of H3K36 establishes transcriptional asymmetry in repair by promoting canonical TC-NER of the transcribed strand (TS) and suppressing cryptic TC-NER of the NTS.

A half century of exploring DNA excision repair in chromatin.

DNA in eukaryotic cells is packaged into the compact and dynamic structure of chromatin. This packaging is a double-edged sword for DNA repair and genomic stability. Chromatin restricts the access of repair proteins to DNA lesions embedded in nucleosomes and higher order chromatin structures. However, chromatin also serves as a signaling platform in which post-translational modifications of histones and other chromatin-bound proteins promote lesion recognition and repair. Similarly, chromatin modulates the formation of DNA damage, promoting or suppressing lesion formation depending on the chromatin context. Therefore, the modulation of DNA damage and its repair in chromatin is crucial to our understanding of the fate of potentially mutagenic and carcinogenic lesions in DNA. Here, we survey many of the landmark findings on DNA damage and repair in chromatin over the last 50 years (i.e., since the beginning of this field), focusing on excision repair, the first repair mechanism studied in the chromatin landscape. For example, we highlight how the impact of chromatin on these processes explains the distinct patterns of somatic mutations observed in cancer genomes.

Distinct roles for RSC and SWI/SNF chromatin remodelers in genomic excision repair.

Nucleosomes are a significant barrier to the repair of UV damage because they impede damage recognition by nucleotide excision repair (NER). The RSC and SWI/SNF chromatin remodelers function in cells to promote DNA access by moving or evicting nucleosomes, and both have been linked to NER in yeast. Here, we report genome-wide repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast cells lacking RSC or SWI/SNF activity. Our data indicate that SWI/SNF is not generally required for NER but instead promotes repair of CPD lesions at specific yeast genes. In contrast, mutation or depletion of RSC subunits causes a general defect in NER across the yeast genome. Our data indicate that RSC is required for repair not only in nucleosomal DNA but also in neighboring linker DNA and nucleosome-free regions (NFRs). Although depletion of the RSC catalytic subunit also affects base excision repair (BER) of N-methylpurine (NMP) lesions, RSC activity is less important for BER in linker DNA and NFRs. Furthermore, our data indicate that RSC plays a direct role in transcription-coupled NER (TC-NER) of transcribed DNA. These findings help to define the specific genomic and chromatin contexts in which each chromatin remodeler functions in DNA repair, and indicate that RSC plays a unique function in facilitating repair by both NER subpathways.

Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process.

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 non-motile complexes or conducts a one-dimensional search via either random diffusion or constrained motion. Atomic force microcopy analysis of Rad4 with the ß-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 ß-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 Repair in Nucleosomes: Insights from Histone Modifications and Mutants.

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.

Asymmetric repair of UV damage in nucleosomes imposes a DNA strand polarity on somatic mutations in skin cancer.

Nucleosomes inhibit excision repair of DNA damage caused by ultraviolet (UV) light, and it has been generally assumed that repair inhibition is equivalent on both sides of the nucleosome dyad. Here, we use genome-wide repair data to show that repair of UV damage in nucleosomes is asymmetric. In yeast, nucleosomes inhibit nucleotide excision repair (NER) of the nontranscribed strand (NTS) of genes in an asymmetric manner, with faster repair of UV damage occurring on the 5' side of the nucleosomal DNA. Analysis of genomic repair data from UV-irradiated human cells indicates that NER activity along the NTS is also elevated on the 5' side of nucleosomes, consistent with the repair asymmetry observed in yeast nucleosomes. Among intergenic nucleosomes, repair activity is elevated on the 5' side of both DNA strands. The distribution of somatic mutations in nucleosomes shows the opposite asymmetry in NER-proficient skin cancers, but not in NER-deficient cancers, indicating that asymmetric repair of nucleosomal DNA imposes a strand polarity on UV mutagenesis. Somatic mutations are enriched on the relatively slow-repairing 3' side of the nucleosomal DNA, particularly at positions where the DNA minor groove faces away from the histone octamer. Asymmetric repair and mutagenesis are likely caused by differential accessibility of the nucleosomal DNA, a consequence of its left-handed wrapping around the histone octamer.

Genome-wide role of Rad26 in promoting transcription-coupled nucleotide excision repair in yeast chromatin.

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion of SPT4 significantly restores TC-NER across the genome in a rad26∆ mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.

Recurrent Noncoding Mutations in Skin Cancers: UV Damage Susceptibility or Repair Inhibition as Primary Driver?

Somatic mutations arising in human skin cancers are heterogeneously distributed across the genome, meaning that certain genomic regions (e.g., heterochromatin or transcription factor binding sites) have much higher mutation densities than others. Regional variations in mutation rates are typically not a consequence of selection, as the vast majority of somatic mutations in skin cancers are passenger mutations that do not promote cell growth or transformation. Instead, variations in DNA repair activity, due to chromatin organization and transcription factor binding, have been proposed to be a primary driver of mutational heterogeneity in melanoma. However, as discussed in this review here, recent studies indicate that chromatin organization and transcription factor binding also significantly modulate the rate at which UV lesions form in DNA. The authors propose that local variations in lesion susceptibility may be an important driver of mutational hotspots in melanoma and other skin cancers, particularly at binding sites for ETS transcription factors.

R-loop formation by dCas9 is mutagenic in Saccharomyces cerevisiae.

Cas9 binds and cleaves specific DNA sequences by inducing the formation of an R-loop between the guide RNA and its genomic target. While targeting of active Cas9 to a genomic locus is highly mutagenic because Cas9 creates DNA double strand breaks, targeting of dead Cas9 (dCas9) is presumed not to be mutagenic, as dCas9 lacks DNA endonuclease activity. Here, we show that dCas9 targeting induces mutations in yeast, particularly when targeted to the non-transcribed strand of a gene. dCas9-induced mutations cluster near the guide RNA target region and are comprised of single nucleotide substitutions, small insertions and deletions, and even complex mutations, depending upon the particular guide RNA target. We show that many of these mutations are a consequence of cytosine deamination events occurring on the non-target strand of the dCas9-induced R-loop, while others are associated with homopolymer instability or translesion DNA synthesis. Targeting of dCas9 by a mismatch-containing guide RNA also increases CAN1 mutation frequency, particularly in an ung1Δ mutant strain, suggesting that dCas9 induces mutations through similar mechanisms at off-target sites. These findings indicate that DNA binding by dCas9 is mutagenic in yeast, likely because dCas9 induces the formation of an R-loop at its target site.

Nucleosome positions establish an extended mutation signature in melanoma.

Ultraviolet (UV) light-induced mutations are unevenly distributed across skin cancer genomes, but the molecular mechanisms responsible for this heterogeneity are not fully understood. Here, we assessed how nucleosome structure impacts the positions of UV-induced mutations in human melanomas. Analysis of mutation positions from cutaneous melanomas within strongly positioned nucleosomes revealed a striking ~10 base pair (bp) oscillation in mutation density with peaks occurring at dinucleotides facing away from the histone octamer. Additionally, higher mutation density at the nucleosome dyad generated an overarching "translational curvature" across the 147 bp of DNA that constitutes the nucleosome core particle. This periodicity and curvature cannot be explained by sequence biases in nucleosomal DNA. Instead, our genome-wide map of UV-induced cyclobutane pyrimidine dimers (CPDs) indicates that CPD formation is elevated at outward facing dinucleotides, mirroring the oscillation of mutation density within nucleosome-bound DNA. Nucleotide excision repair (NER) activity, as measured by XR-seq, inversely correlated with the curvature of mutation density associated with the translational setting of the nucleosome. While the 10 bp periodicity of mutations is maintained across nucleosomes regardless of chromatin state, histone modifications, and transcription levels, overall mutation density and curvature across the core particle increased with lower transcription levels. Our observations suggest structural conformations of DNA promote CPD formation at specific sites within nucleosomes, and steric hindrance progressively limits lesion repair towards the nucleosome dyad. Both mechanisms create a unique extended mutation signature within strongly positioned nucleosomes across the human genome.

Genome-wide maps of alkylation damage, repair, and mutagenesis in yeast reveal mechanisms of mutational heterogeneity.

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.

A cassette of basic amino acids in histone H2B regulates nucleosome dynamics and access to DNA damage.

Nucleosome dynamics, such as spontaneous DNA unwrapping, are postulated to have a critical role in regulating the access of DNA repair machinery to DNA lesions within nucleosomes. However, the specific histone domains that regulate nucleosome dynamics and the impact of such changes in intrinsic nucleosome dynamics on DNA repair are not well understood. Previous studies identified a highly conserved region in the N-terminal tail of histone H2B known as the histone H2Brepression (or HBR) domain, which has a significant influence on gene expression, chromatin assembly, and DNA damage formation and repair. However, the molecular mechanism(s) that may account for these observations are limited. In this study, we characterized the stability and dynamics of ΔHBR mutant nucleosome core particles (NCPs) in vitro by restriction enzyme accessibility (REA), FRET, and temperature-induced sliding of histone octamers. Our results indicate that ΔHBR-NCPs are more dynamic, with a larger steady-state fraction of the NCP population occupying the unwrapped state than for WT-NCPs. Additionally, ΔHBR-histone octamers are more susceptible to temperature-induced sliding on DNA than WT histone octamers. Furthermore, we show that the activity of base excision repair enzymes at uracil lesions and single nucleotide gaps is enhanced in a site-specific manner in ΔHBR-NCPs. This enhanced activity correlates well with regions exhibiting increased DNA unwrapping. Finally, removal of the HBR domain is not sufficient to completely alleviate the structural constraints imposed by histone octamers on the activity of base excision repair enzymes.

Chromosomal landscape of UV damage formation and repair at single-nucleotide resolution.

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 DNA-bound 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.

A basic domain in the histone H2B N-terminal tail is important for nucleosome assembly by FACT.

Nucleosome assembly in vivo requires assembly factors, such as histone chaperones, to bind to histones and mediate their deposition onto DNA. In yeast, the essential histone chaperone FACT (FAcilitates Chromatin Transcription) functions in nucleosome assembly and H2A-H2B deposition during transcription elongation and DNA replication. Recent studies have identified candidate histone residues that mediate FACT binding to histones, but it is not known which histone residues are important for FACT to deposit histones onto DNA during nucleosome assembly. In this study, we report that the histone H2B repression (HBR) domain within the H2B N-terminal tail is important for histone deposition by FACT. Deletion of the HBR domain causes significant defects in histone occupancy in the yeast genome, particularly at HBR-repressed genes, and a pronounced increase in H2A-H2B dimers that remain bound to FACT in vivo Moreover, the HBR domain is required for purified FACT to efficiently assemble recombinant nucleosomes in vitro We propose that the interaction between the highly basic HBR domain and DNA plays an important role in stabilizing the nascent nucleosome during the process of histone H2A-H2B deposition by FACT.

Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge.

Nucleosomes affect Cas9 binding and activity at on-target sites, but their impact at off-target sites is unknown. To investigate how nucleosomes affect Cas9 cleavage at off-target sites in vitro, we used a single guide RNA (sgRNA) that has been previously shown to efficiently direct Cas9 cleavage at the edge of the strongly positioned 601 nucleosome. Our data indicate that single mismatches between the sgRNA and DNA target have relatively little effect on Cas9 cleavage of naked DNA substrates, but strongly inhibit cleavage of nucleosome substrates, particularly when the mismatch is in the sgRNA "seed" region. These findings indicate that nucleosomes may enhance Cas9 specificity by inhibiting cleavage of off-target sites at the nucleosome edge.

Site-specific Acetylation of Histone H3 Decreases Polymerase ß Activity on Nucleosome Core Particles in Vitro.

Histone posttranslational modifications have been associated with changes in chromatin structure necessary for transcription, replication, and DNA repair. Acetylation is one of the most studied and best characterized histone posttranslational modifications, but it is not known if histone acetylation modulates base excision repair of DNA lesions in chromatin. To address this question, we generated nucleosome core particles (NCPs) containing site-specifically acetylated H3K14 or H3K56 and measured repair of uracil and single-nucleotide gaps. We find that H3K56Ac and H3K14Ac do not significantly contribute to removal of uracils by uracil DNA glycosylase regardless of the translational or rotational position of the lesions within NCPs. In repair of single-nucleotide gaps, however, the presence of H3K56Ac or H3K14Ac in NCPs decreases the gap-filling activity of DNA polymerase ß near the dyad center, with H3K14Ac exhibiting stronger inhibition. To a lesser extent, H3K56Ac induces a similar effect near the DNA ends. Moreover, using restriction enzyme accessibility, we detect no changes in NCP structure or dynamics between H3K14Ac-NCPs and WT-NCPs containing single-nucleotide gaps. Thus, acetylation at H3K56 and H3K14 in nucleosomes may promote alternative gap-filling pathways by inhibiting DNA polymerase ß activity.

The amino-terminal tails of histones H2A and H3 coordinate efficient base excision repair, DNA damage signaling and postreplication repair in Saccharomyces cerevisiae.

Histone amino-terminal tails (N-tails) are required for cellular resistance to DNA damaging agents; therefore, we examined the role of histone N-tails in regulating DNA damage response pathways in Saccharomyces cerevisiae. Combinatorial deletions reveal that the H2A and H3 N-tails are important for the removal of MMS-induced DNA lesions due to their role in regulating the basal and MMS-induced expression of DNA glycosylase Mag1. Furthermore, overexpression of Mag1 in a mutant lacking the H2A and H3 N-tails rescues base excision repair (BER) activity but not MMS sensitivity. We further show that the H3 N-tail functions in the Rad9/Rad53 DNA damage signaling pathway, but this function does not appear to be the primary cause of MMS sensitivity of the double tailless mutants. Instead, epistasis analyses demonstrate that the tailless H2A/H3 phenotypes are in the RAD18 epistasis group, which regulates postreplication repair. We observed increased levels of ubiquitylated PCNA and significantly lower mutation frequency in the tailless H2A/H3 mutant, indicating a defect in postreplication repair. In summary, our data identify novel roles of the histone H2A and H3 N-tails in (i) regulating the expression of a critical BER enzyme (Mag1), (ii) supporting efficient DNA damage signaling and (iii) facilitating postreplication repair.