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.

Genome-wide impact of cytosine methylation and DNA sequence context on UV-induced CPD formation.

Exposure to ultraviolet (UV) light is the primary etiological agent for skin cancers because UV damages cellular DNA. The most frequent form of UV damage is the cyclobutane pyrimidine dimer (CPD), which consists of covalent linkages between neighboring pyrimidine bases in DNA. In human cells, the 5' position of cytosine bases in CG dinucleotides is frequently methylated, and methylated cytosines in the TP53 tumor suppressor are often sites of mutation hotspots in skin cancers. It has been argued that this is because cytosine methylation promotes UV-induced CPD formation; however, the effects of cytosine methylation on CPD formation are controversial, with conflicting results from previous studies. Here, we use a genome-wide method known as CPD-seq to map UVB- and UVC-induced CPDs across the yeast genome in the presence or absence in vitro methylation by the CpG methyltransferase M.SssI. Our data indicate that cytosine methylation increases UVB-induced CPD formation nearly 2-fold relative to unmethylated DNA, but the magnitude of induction depends on the flanking sequence context. Sequence contexts with a 5' guanine base (e.g., GCCG and GTCG) show the strongest induction due to cytosine methylation, potentially because these sequence contexts are less efficient at forming CPD lesions in the absence of methylation. We show that cytosine methylation also modulates UVC-induced CPD formation, albeit to a lesser extent than UVB. These findings can potentially reconcile previous studies, and define the impact of cytosine methylation on UV damage across a eukaryotic genome.

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.

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.

Detecting recurrent passenger mutations in melanoma by targeted UV damage sequencing.

Sequencing of melanomas has identified hundreds of recurrent mutations in both coding and non-coding DNA. These include a number of well-characterized oncogenic driver mutations, such as coding mutations in the BRAF and NRAS oncogenes, and non-coding mutations in the promoter of telomerase reverse transcriptase (TERT). However, the molecular etiology and significance of most of these mutations is unknown. Here, we use a new method known as CPD-capture-seq to map UV-induced cyclobutane pyrimidine dimers (CPDs) with high sequencing depth and single nucleotide resolution at sites of recurrent mutations in melanoma. Our data reveal that many previously identified drivers and other recurrent mutations in melanoma occur at CPD hotspots in UV-irradiated melanocytes, often associated with an overlapping binding site of an E26 transformation-specific (ETS) transcription factor. In contrast, recurrent mutations in the promoters of a number of known or suspected cancer genes are not associated with elevated CPD levels. Our data indicate that a subset of recurrent protein-coding mutations are also likely caused by ETS-induced CPD hotspots. This analysis indicates that ETS proteins profoundly shape the mutation landscape of melanoma and reveals a method for distinguishing potential driver mutations from passenger mutations whose recurrence is due to elevated UV damage.

Genome-wide maps of UVA and UVB mutagenesis in yeast reveal distinct causative lesions and mutational strand asymmetries.

Ultraviolet (UV) light primarily causes C > T substitutions in lesion-forming dipyrimidine sequences. However, many of the key driver mutations in melanoma do not fit this canonical UV signature, but are instead caused by T > A, T > C, or C > A substitutions. To what extent exposure to the UVB or UVA spectrum of sunlight can induce these noncanonical mutation classes, and the molecular mechanism involved is unclear. Here, we repeatedly exposed wild-type or repair-deficient yeast (Saccharomyces cerevisiae) to UVB or UVA light and characterized the resulting mutations by whole genome sequencing. Our data indicate that UVB induces C > T and T > C substitutions in dipyrimidines, and T > A substitutions that are often associated with thymine-adenine (TA) sequences. All of these mutation classes are induced in nucleotide excision repair-deficient cells and show transcriptional strand asymmetry, suggesting they are caused by helix-distorting UV photoproducts. In contrast, UVA exposure induces orders of magnitude fewer mutations with a distinct mutation spectrum. UVA-induced mutations are elevated in Ogg1-deficient cells, and the resulting spectrum consists almost entirely of C > A/G > T mutations, indicating they are likely derived from oxidative guanine lesions. These mutations show replication asymmetry, with elevated G > T mutations on the leading strand, suggesting there is a strand bias in the removal or bypass of guanine lesions during replication. Finally, we develop a mutation reporter to show that UVA induces a G > T reversion mutation in yeast that mimics the oncogenic NRAS Q61K mutation in melanoma. Taken together, these findings indicate that UVA and UVB exposure can induce many of the noncanonical mutation classes that cause driver mutations in melanoma.

Contributions of replicative and translesion DNA polymerases to mutagenic bypass of canonical and atypical UV photoproducts.

UV exposure induces a mutation signature of C > T substitutions at dipyrimidines in skin cancers. We recently identified additional UV-induced AC > TT and A > T substitutions that could respectively cause BRAF V600K and V600E oncogenic mutations. The mutagenic bypass mechanism past these atypical lesions, however, is unknown. Here, we whole genome sequenced UV-irradiated yeast and used reversion reporters to delineate the roles of replicative and translesion DNA polymerases in mutagenic bypass of UV-lesions. Our data indicates that yeast DNA polymerase eta (pol η) has varied impact on UV-induced mutations: protecting against C > T substitutions, promoting T > C and AC > TT substitutions, and not impacting A > T substitutions. Surprisingly, deletion rad30Δ increased novel UV-induced C > A substitutions at CA dinucleotides. In contrast, DNA polymerases zeta (pol ζ) and epsilon (pol ε) participated in AC > TT and A > T mutations. These results uncover lesion-specific accurate and mutagenic bypass of UV lesions, which likely contribute to key driver mutations in melanoma.

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.

Molecular mechanism of UV damage modulation in nucleosomes.

Exposure to ultraviolet (UV) light causes the formation of mutagenic cyclobutane pyrimidine dimers (CPDs) in cellular DNA. Previous studies have revealed that CPD formation in nucleosomes, the building blocks of chromatin, shows a striking ∼10 base pair (bp) periodic pattern. CPD formation is suppressed at positions where the DNA minor groove faces toward the histone octamer (minor-in) and elevated CPD formation at positions where the minor groove faces away from the histone octamer (minor-out). However, the molecular mechanism underlying this nucleosome photofootprint is unclear. Here, we analyzed ∼180 high-resolution nucleosome structures to characterize whether differences in DNA mobility or conformation are responsible for the CPD modulation in nucleosomes. Our results indicate that differences in DNA mobility cannot explain CPD modulation in nucleosome. Instead, we find that the sharp DNA bending around the histone octamer results in DNA conformations with structural parameters more susceptible to UV damage formation at minor-out positions and more resistant to CPD formation at minor-in positions. This analysis reveals the molecular mechanism responsible for periodic modulation of CPD formation and UV mutagenesis in nucleosomal DNA.

Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae.

The versatility of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) genome editing makes it a popular tool for many research and biotechnology applications. Recent advancements in genome editing in eukaryotic organisms, like fungi, allow for precise manipulation of genetic information and fine-tuned control of gene expression. Here, we provide an overview of CRISPR genome editing technologies in yeast, with a particular focus on Saccharomyces cerevisiae. We describe the tools and methods that have been previously developed for genome editing in Saccharomyces cerevisiae and discuss tips and experimental tricks for promoting efficient, marker-free genome editing in this model organism. These include sgRNA design and expression, multiplexing genome editing, optimizing Cas9 expression, allele-specific editing in diploid cells, and understanding the impact of chromatin on genome editing. Finally, we summarize recent studies describing the potential pitfalls of using CRISPR genome targeting in yeast, including the induction of background mutations.

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.

High-resolution mapping demonstrates inhibition of DNA excision repair by transcription factors.

DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method, N-methylpurine-sequencing (NMP-seq), and characterized alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data show that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs ARS binding factor 1 (Abf1) and rDNA enhancer binding protein 1 (Reb1), but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro, causing slow repair both within the core motif and its adjacent DNA. Repair of ultraviolet (UV) damage by nucleotide excision repair (NER) was also inhibited by TF binding. Interestingly, TF binding inhibits a larger DNA region for NER relative to BER. The observed effects are caused by the TF-DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.

Mapping atypical UV photoproducts in vitro and across the S. cerevisiae genome.

Exposure to ultraviolet (UV) light induces DNA damage, predominantly cyclobutane pyrimidine dimers (CPD) and 6,4-photoproducts (6,4-PP), as well as rare, atypical photoproducts at thymidine-adenine (TA) sequences. We have recently shown 'TA' photoproducts are induced in UV-irradiated oligonucleotides and across the budding yeast genome. Here, we describe a protocol for mapping atypical 'TA' photoproducts in vitro and in vivo. This protocol overcomes the technical challenges involved in accurately mapping such rare photoproducts by using ultraviolet damage endonuclease (UVDE) enzymes. For complete details on the use and execution of this protocol, please refer to Laughery et al. (2020).

dCas9 binding inhibits the initiation of base excision repair in vitro.

Cas9 targets DNA during genome editing by forming an RNA:DNA heteroduplex (R-loop) between the Cas9-bound guide RNA and the targeted DNA strand. We have recently demonstrated that R-loop formation by catalytically inactive Cas9 (dCas9) is inherently mutagenic, in part, by promoting spontaneous cytosine deamination within the non-targeted single-stranded DNA of the dCas9-induced R-loop. However, the extent to which dCas9 binding and R-loop formation affect the subsequent repair of uracil lesions or other damaged DNA bases is unclear. Here, we show that DNA binding by dCas9 inhibits initiation of base excision repair (BER) for uracil lesions in vitro. Our data indicate that cleavage of uracil lesions by Uracil-DNA glycosylase (UDG) is generally inhibited at dCas9-bound DNA, in both the dCas9:sgRNA-bound target strand (TS) or the single-stranded non-target strand (NT). However, cleavage of a uracil lesion within the base editor window of the NT strand was less inhibited than at other locations, indicating that this site is more permissive to UDG activity. Furthermore, our data suggest that dCas9 binding to PAM sites can inhibit UDG activity. However, this non-specific inhibition can be relieved with the addition of an sgRNA lacking sequence complementarity to the DNA substrate. Moreover, we show that dCas9 binding also inhibits human single-strand selective monofunctional uracil-DNA glycosylase (SMUG1). Structural analysis of a Cas9-bound target site subsequently suggests a molecular mechanism for BER inhibition. Taken together, our results imply that dCas9 (or Cas9) binding may promote background mutagenesis by inhibiting the removal of DNA base lesions by BER.

Damage mapping techniques and the light they have shed on canonical and atypical UV photoproducts.

Ultraviolet (UV) light is a pervasive threat to the DNA of terrestrial organisms. UV light induces helix-distorting DNA lesions, primarily cyclobutane pyrimidine dimers (CPDs) that form between neighboring pyrimidine bases. Unrepaired CPD lesions cause cytosine-to-thymine (C>T) substitutions in dipyrimidine sequences, which is the predominant mutation class in skin cancer genomes. However, many driver mutations in melanoma (e.g., in the BRAF and NRAS oncogenes) do not fit this UV mutation signature. Recent studies have brought to light the intriguing hypothesis that these driver mutations may be induced by infrequent or atypical UV photoproducts, including pyrimidine 6-4 pyrimidone photoproducts (6-4PP) and thymine-adenine (TA) photoproducts. Here, we review innovative methods for mapping both canonical and atypical UV-induced photoproducts across the genome.

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.

Mutperiod: Analysis of periodic mutation rates in nucleosomes.

Nucleosomes modulate DNA damage and repair, resulting in periodic mutation rates in nucleosomal DNA. Previous research has characterized these patterns in many sequenced tumor genomes; however, computational tools to identify and quantify these periodicities have not been developed for the broader scientific community. Here, we describe mutperiod, a Python and R based toolset that quantifies nucleosome mutational periodicities and compares them across different genetic and cellular backgrounds. We use mutperiod to demonstrate that DNA mismatch repair contributes to the nucleosome mutational periodicity observed in esophageal adenocarcinomas, and that the strength of this mutational periodicity varies in different chromatin states.

Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability.

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.

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.