Mutations of the circadian clock genes Cry, Per, or Bmal1 have different effects on the transcribed and nontranscribed strands of cycling genes.

One important goal of circadian medicine is to apply time-of-day dosing to improve the efficacy of chemotherapy. However, limited knowledge of how the circadian clock regulates DNA repair presents a challenge to mechanism-based clinical application. We studied time-series genome-wide nucleotide excision repair in liver and kidney of wild type and three different clock mutant genotypes (Cry1-/-Cry2-/-, Per1-/-Per2-/-, and Bmal1-/-). Rhythmic repair on the nontranscribed strand was lost in all three clock mutants. Conversely, rhythmic repair of hundreds of genes on the transcribed strand (TSs) persisted in the livers of Cry1-/-Cry2-/- and Per1-/-Per2-/- mice. We identified a tissue-specific, promoter element-driven repair mode on TSs of collagen and angiogenesis genes in the absence of clock activators or repressors. Furthermore, repair on TSs of thousands of genes was altered when the circadian clock is disrupted. These data contribute to a better understanding of the regulatory role of the circadian clock on nucleotide excision repair in mammals and may be invaluable toward the design of time-aware platinum-based interventions in cancer.

Global repair is the primary nucleotide excision repair subpathway for the removal of pyrimidine-pyrimidone (6-4) damage from the Arabidopsis genome.

Ultraviolet (UV) component of solar radiation impairs genome stability by inducing the formation of pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] in plant genomes. (6-4)PPs disrupt growth and development by interfering with transcription and DNA replication. To resist UV stress, plants employ both photoreactivation and nucleotide excision repair that excises oligonucleotide containing (6-4)PPs through two subpathways: global and transcription-coupled excision repair (TCR). Here, we analyzed the genome-wide excision repair-mediated repair of (6-4)PPs in Arabidopsis thaliana and found that (6-4)PPs can be repaired by TCR; however, the main subpathway to remove (6-4)PPs from the genome is global repair. Our analysis showed that open chromatin genome regions are more rapidly repaired than heterochromatin regions, and the repair level peaks at the promoter, transcription start site and transcription end site of genes. Our study revealed that the repair of (6-4)PP in plants showed a distinct genome-wide repair profile compared to the repair of other major UV-induced DNA lesion called cyclobutane pyrimidine dimers (CPDs).

Cross-species investigation into the requirement of XPA for nucleotide excision repair.

After reconstitution of nucleotide excision repair (excision repair) with XPA, RPA, XPC, TFIIH, XPF-ERCC1 and XPG, it was concluded that these six factors are the minimal essential components of the excision repair machinery. All six factors are highly conserved across diverse organisms spanning yeast to humans, yet no identifiable homolog of the XPA gene exists in many eukaryotes including green plants. Nevertheless, excision repair is reported to be robust in the XPA-lacking organism, Arabidopsis thaliana, which raises a fundamental question of whether excision repair could occur without XPA in other organisms. Here, we performed a phylogenetic analysis of XPA across all species with annotated genomes and then quantitatively measured excision repair in the absence of XPA using the sensitive whole-genome qXR-Seq method in human cell lines and two model organisms, Caenorhabditis elegans and Drosophila melanogaster. We find that although the absence of XPA results in inefficient excision repair and UV-sensitivity in humans, flies, and worms, excision repair of UV-induced DNA damage is detectable over background. These studies have yielded a significant discovery regarding the evolution of XPA protein and its mechanistic role in nucleotide excision repair.

Genome-wide transcription and repair maps of Caenorhabditis elegans.

We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts in the Caenorhabditis elegans (C. elegans) genome. We focus on the C. elegans ortholog of the human XPC-deficient strain (xpc-1) and its exclusive use of transcription-coupled repair. We provide evidence demonstrating the utility of xpc-1 XR-seq as a remarkable tool for detecting nascent transcription and identifying new transcripts. The integration of epigenetic markers, chromatin states, enhancer RNA and long intergenic non-coding RNA annotations supports the robust detection of intergenic nascent transcription by XR-seq. Overall, our results provide a comprehensive view of the transcription-coupled repair landscape in C. elegans, highlighting its potential contributions to our understanding of DNA repair mechanisms and non-coding RNA biology.

In vitro DNA repair genomics using XR-seq with Escherichia coli and mammalian cell-free extracts.

The XR-seq (eXcision Repair-sequencing) method has been extensively used to map nucleotide excision repair genome-wide in organisms ranging from Escherichia coli to yeast, Drosophila, Arabidopsis, mice, and humans. The basic feature of the method is to capture the excised oligomers carrying DNA damage, sequence them, and align their sequences to the genome. We wished to perform XR-seq in vitro with cell-free extract supplemented with a damaged DNA substrate so as to have greater flexibility in investigating factors that affect nucleotide excision repair in the cellular context [M. J. Smerdon, J. J. Wyrick, S. Delaney, J. Biol. Chem. 299, 105118 (2023)]. We report here the successful use of ultraviolet light-irradiated plasmids as substrates for repair in vitro and in vivo by E. coli and E. coli cell-free extracts and by mammalian cell-free extract. XR-seq analyses demonstrated common excision product length and sequence characteristics in vitro and in vivo for both the bacterial and mammalian systems. This approach is expected to help understand the effects of epigenetics and other cellular factors and conditions on DNA repair.

Nucleotide Excision Repair of Aflatoxin-induced DNA Damage within the 3D Human Genome Organization.

Aflatoxin B1 (AFB1), a potent mycotoxin, is one of the two primary risk factors that cause liver cancer. In the liver, the bioactivated AFB1 intercalates into the DNA double helix to form a bulky DNA adduct which will lead to mutation if left unrepaired. We have adapted the tXR-seq method to measure the nucleotide excision repair of AFB1-induced DNA adducts. We have found that transcription-coupled repair plays a major role in the damage removal process and the released excision products have a distinctive length distribution pattern. We further analyzed the impact of 3D genome organization on the repair of AFB1-induced DNA adducts. We have revealed that chromosomes close to the nuclear center and A compartments undergo expedited repair processes. Notably, we observed an accelerated repair around both TAD boundaries and loop anchors. These findings provide insights into the complex interplay between repair, transcription, and 3D genome organization, shedding light on the mechanisms underlying AFB1-induced liver cancer.

Preservation of circadian rhythm in hepatocellular cancer.

Circadian rhythms are controlled at the cellular level by a molecular clock consisting of several genes/proteins engaged in a transcription-translation-degradation feedback loop. These core clock proteins regulate thousands of tissue-specific genes. Regarding circadian control in neoplastic tissues, reports to date have demonstrated anomalous circadian function in tumor models and cultured tumor cells. We have extended these studies by analyzing circadian rhythmicity genome-wide in a mouse model of liver cancer, in which mice treated with diethylnitrosamine at 15 days develop liver tumors by 6 months. We injected tumor-bearing and control tumor-free mice with cisplatin every 2 h over a 24-h cycle; 2 h after each injection mice were sacrificed and gene expression was measured by XR-Seq (excision repair sequencing) assay. Rhythmic expression of several core clock genes was observed in both healthy liver and tumor, with clock genes in tumor exhibiting typically robust amplitudes and a modest phase advance. Interestingly, although normal hepatic cells and hepatoma cancer cells expressed a comparable number of genes with circadian rhythmicity (clock-controlled genes), there was only about 10% overlap between the rhythmic genes in normal and cancerous cells. "Rhythmic in tumor only" genes exhibited peak expression times mainly in daytime hours, in contrast to the more common pre-dawn and pre-dusk expression times seen in healthy livers. Differential expression of genes in tumors and healthy livers across time may present an opportunity for more efficient anticancer drug treatment as a function of treatment time.

Nucleotide excision repair in Human cell lines lacking both XPC and CSB proteins.

Nucleotide excision repair removes UV-induced DNA damage through two distinct sub-pathways, global repair and transcription-coupled repair (TCR). Numerous studies have shown that in human and other mammalian cell lines that the XPC protein is required for repair of DNA damage from nontranscribed DNA via global repair and the CSB protein is required for repair of lesions from transcribed DNA via TCR. Therefore, it is generally assumed that abrogating both sub-pathways with an XPC-/-/CSB-/- double mutant would eliminate all nucleotide excision repair. Here we describe the construction of three different XPC-/-/CSB-/- human cell lines that, contrary to expectations, perform TCR. The XPC and CSB genes were mutated in cell lines derived from Xeroderma Pigmentosum patients as well as from normal human fibroblasts and repair was analyzed at the whole genome level using the very sensitive XR-seq method. As predicted, XPC-/- cells exhibited only TCR and CSB-/- cells exhibited only global repair. However, the XPC-/-/CSB-/- double mutant cell lines, although having greatly reduced repair, exhibited TCR. Mutating the CSA gene to generate a triple mutant XPC-/-/CSB-/-/CSA-/- cell line eliminated all residual TCR activity. Together, these findings provide new insights into the mechanistic features of mammalian nucleotide excision repair.

Molecular Mechanisms of Transcription-Coupled Repair.

Transcription-coupled repair (TCR), discovered as preferential nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers located in transcribed mammalian genes compared to those in nontranscribed regions of the genome, is defined as faster repair of the transcribed strand versus the nontranscribed strand in transcribed genes. The phenomenon, universal in model organisms including Escherichia coli, yeast, Arabidopsis, mice, and humans, involves a translocase that interacts with both RNA polymerase stalled at damage in the transcribed strand and nucleotide excision repair proteins to accelerate repair. Drosophila, a notable exception, exhibits TCR but lacks an obvious TCR translocase. Mutations inactivating TCR genes cause increased damage-induced mutagenesis in E. coli and severe neurological and UV sensitivity syndromes in humans. To date, only E. coli TCR has been reconstituted in vitro with purified proteins. Detailed investigations of TCR using genome-wide next-generation sequencing methods, cryo-electron microscopy, single-molecule analysis, and other approaches have revealed fascinating mechanisms.

The Mfd protein is the transcription-repair coupling factor (TRCF) in Mycobacterium smegmatis.

In vitro and in vivo experiments with Escherichia coli have shown that the Mfd translocase is responsible for transcription-coupled repair, a subpathway of nucleotide excision repair involving the faster rate of repair of the transcribed strand than the nontranscribed strand. Even though the mfd gene is conserved in all bacterial lineages, there is only limited information on whether it performs the same function in other bacterial species. Here, by genome scale analysis of repair of UV-induced cyclobutane pyrimidine dimers, we find that the Mfd protein is the transcription-repair coupling factor in Mycobacterium smegmatis. This finding, combined with the inverted strandedness of UV-induced mutations in WT and mfd-E. coli and Bacillus subtilis indicate that the Mfd protein is the universal transcription-repair coupling factor in bacteria.

Analysis of mammalian circadian clock protein complexes over a circadian cycle.

Circadian rhythmicity is maintained by a set of core clock proteins including the transcriptional activators CLOCK and BMAL1, and the repressors PER (PER1, PER2, and PER3), CRY (CRY1 and CRY2), and CK1δ. In mice, peak expression of the repressors in the early morning reduces CLOCK- and BMAL1-mediated transcription/translation of the repressors themselves. By late afternoon the repressors are largely depleted by degradation, and thereby their expression is reactivated in a cycle repeated every 24 h. Studies have characterized a variety of possible protein interactions and complexes associated with the function of this transcription-translation feedback loop. Our prior investigation suggested there were two circadian complexes responsible for rhythmicity, one containing CLOCK-BMAL and the other containing PER2, CRY1, and CK1δ. In this investigation, we acquired data from glycerol gradient centrifugation and gel filtration chromatography of mouse liver extracts obtained at different circadian times to further characterize circadian complexes. In addition, anti-PER2 and anti-CRY1 immunoprecipitates obtained from the same extracts were analyzed by liquid chromatography-tandem mass spectrometry to identify components of circadian complexes. Our results confirm the presence of discrete CLOCK-BMAL1 and PER-CRY-CK1δ complexes at the different circadian time points, provide masses of 255 and 707 kDa, respectively, for these complexes, and indicate that these complexes are composed principally of the core circadian proteins.

Effects of replication domains on genome-wide UV-induced DNA damage and repair.

Nucleotide excision repair is the primary repair mechanism that removes UV-induced DNA lesions in placentals. Unrepaired UV-induced lesions could result in mutations during DNA replication. Although the mutagenesis of pyrimidine dimers is reasonably well understood, the direct effects of replication fork progression on nucleotide excision repair are yet to be clarified. Here, we applied Damage-seq and XR-seq techniques and generated replication maps in synchronized UV-treated HeLa cells. The results suggest that ongoing replication stimulates local repair in both early and late replication domains. Additionally, it was revealed that lesions on lagging strand templates are repaired slower in late replication domains, which is probably due to the imbalanced sequence context. Asymmetric relative repair is in line with the strand bias of melanoma mutations, suggesting a role of exogenous damage, repair, and replication in mutational strand asymmetry.

Nucleotide excision repair removes thymidine analog 5-ethynyl-2'-deoxyuridine from the mammalian genome.

Nucleotide excision repair is the principal mechanism for removing bulky DNA adducts from the mammalian genome, including those induced by environmental carcinogens such as UV radiation, and anticancer drugs such as cisplatin. Surprisingly, we found that the widely used thymidine analog EdU is a substrate for excision repair when incorporated into the DNA of replicating cells. A number of thymidine analogs were tested, and only EdU was a substrate for excision repair. EdU excision was absent in repair-deficient cells, and in vitro, DNA duplexes bearing EdU were also substrates for excision by mammalian cell-free extracts. We used the excision repair sequencing (XR-seq) method to map EdU repair in the human genome at single-nucleotide resolution and observed that EdU was excised throughout the genome and was subject to transcription-coupled repair as evidenced by higher repair rates in the transcribed strand (TS) relative to the nontranscribed strand (NTS) in transcriptionally active genes. These properties of EdU, combined with its cellular toxicity and ability to cross the blood-brain barrier, make it a potential candidate for treating cancers of the brain, a tissue that typically demonstrates limited replication in adults.

A new technique for genome-wide mapping of nucleotide excision repair without immunopurification of damaged DNA.

Nucleotide excision repair functions to protect genome integrity, and ongoing studies using excision repair sequencing (XR-seq) have contributed to our understanding of how cells prioritize repair across the genome. In this method, the products of excision repair bearing damaged DNA are captured, sequenced, and then mapped genome-wide at single-nucleotide resolution. However, reagent requirements and complex procedures have limited widespread usage of this technique. In addition to the expense of these reagents, it has been hypothesized that the immunoprecipitation step using antibodies directed against damaged DNA may introduce bias in different sequence contexts. Here, we describe a newly developed adaptation called dA-tailing and adaptor ligation (ATL)-XR-seq, a relatively simple XR-seq method that avoids the use of immunoprecipitation targeting damaged DNA. ATL-XR-seq captures repair products by 3'-dA-tailing and 5'-adapter ligation instead of the original 5'- and 3'-dual adapter ligation. This new approach avoids adapter dimer formation during subsequent PCR, omits inefficient and time-consuming purification steps, and is very sensitive. In addition, poly(dA) tail length heterogeneity can serve as a molecular identifier, allowing more repair hotspots to be mapped. Importantly, a comparison of both repair mapping methods showed that no major bias is introduced by the anti-UV damage antibodies used in the original XR-seq procedure. Finally, we also coupled the described dA-tailing approach with quantitative PCR in a new method to quantify repair products. These new methods provide powerful and user-friendly tools to qualitatively and quantitatively measure excision repair.

CSB-independent, XPC-dependent transcription-coupled repair in Drosophila.

Drosophila melanogaster has been extensively used as a model system to study ionizing radiation and chemical-induced mutagenesis, double-strand break repair, and recombination. However, there are only limited studies on nucleotide excision repair in this important model organism. An early study reported that Drosophila lacks the transcription-coupled repair (TCR) form of nucleotide excision repair. This conclusion was seemingly supported by the Drosophila genome sequencing project, which revealed that Drosophila lacks a homolog to CSB, which is known to be required for TCR in mammals and yeasts. However, by using excision repair sequencing (XR-seq) genome-wide repair mapping technology, we recently found that the Drosophila S2 cell line performs TCR comparable to human cells. Here, we have extended this work to Drosophila at all its developmental stages. We find TCR takes place throughout the life cycle of the organism. Moreover, we find that in contrast to humans and other multicellular organisms previously studied, the XPC repair factor is required for both global and transcription-coupled repair in Drosophila.

Genome-wide Excision Repair Map of Cyclobutane Pyrimidine Dimers in Arabidopsis and the Roles of CSA1 and CSA2 Proteins in Transcription-coupled Repair.

Plants depend on light for energy production. However, the UV component in sunlight also inflicts DNA damage, mostly in the form of cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine-pyrimidone photoproducts, which are mutagenic and lethal to the plant cells. These lesions are repaired by blue-light-dependent photolyases and the nucleotide excision repair enzymatic systems. Here, we characterize nucleotide excision repair in Arabidopsis thaliana genome-wide and at single nucleotide resolution with special focus on transcription-coupled repair and the role of the CSA1 and CSA2 genes/proteins in dictating the efficiency and the strand preference of repair of transcribed genes. We demonstrate that CSA1 is the dominant protein in coupling repair to transcription with minor contribution from CSA2.

Genome-wide analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine at single-nucleotide resolution unveils reduced occurrence of oxidative damage at G-quadruplex sites.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (OG), one of the most common oxidative DNA damages, causes genome instability and is associated with cancer, neurological diseases and aging. In addition, OG and its repair intermediates can regulate gene transcription, and thus play a role in sensing cellular oxidative stress. However, the lack of methods to precisely map OG has hindered the study of its biological roles. Here, we developed a single-nucleotide resolution OG-sequencing method, named CLAPS-seq (Chemical Labeling And Polymerase Stalling Sequencing), to measure the genome-wide distribution of both exogenous and endogenous OGs with high specificity. Our data identified decreased OG occurrence at G-quadruplexes (G4s), in association with underrepresentation of OGs in promoters which have high GC content. Furthermore, we discovered that potential quadruplex sequences (PQSs) were hotspots of OGs, implying a role of non-G4-PQSs in OG-mediated oxidative stress response.

Comparative analyses of two primate species diverged by more than 60 million years show different rates but similar distribution of genome-wide UV repair events.

BACKGROUND: Nucleotide excision repair is the primary DNA repair mechanism that removes bulky DNA adducts such as UV-induced pyrimidine dimers. Correspondingly, genome-wide mapping of nucleotide excision repair with eXcision Repair sequencing (XR-seq), provides comprehensive profiling of DNA damage repair. A number of XR-seq experiments at a variety of conditions for different damage types revealed heterogenous repair in the human genome. Although human repair profiles were extensively studied, how repair maps vary between primates is yet to be investigated. Here, we characterized the genome-wide UV-induced damage repair in gray mouse lemur, Microcebus murinus, in comparison to human. RESULTS: We derived fibroblast cell lines from mouse lemur, exposed them to UV irradiation, and analyzed the repair events genome-wide using the XR-seq protocol. Mouse lemur repair profiles were analyzed in comparison to the equivalent human fibroblast datasets. We found that overall UV sensitivity, repair efficiency, and transcription-coupled repair levels differ between the two primates. Despite this, comparative analysis of human and mouse lemur fibroblasts revealed that genome-wide repair profiles of the homologous regions are highly correlated, and this correlation is stronger for highly expressed genes. With the inclusion of an additional XR-seq sample derived from another human cell line in the analysis, we found that fibroblasts of the two primates repair UV-induced DNA lesions in a more similar pattern than two distinct human cell lines do. CONCLUSION: Our results suggest that mouse lemurs and humans, and possibly primates in general, share a homologous repair mechanism as well as genomic variance distribution, albeit with their variable repair efficiency. This result also emphasizes the deep homologies of individual tissue types across the eukaryotic phylogeny.

Circadian clock, carcinogenesis, chronochemotherapy connections.

The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.

The Transcription-Repair Coupling Factor Mfd Prevents and Promotes Mutagenesis in a Context-Dependent Manner.

The mfd (mutation frequency decline) gene was identified by screening an auxotrophic Escherichia coli strain exposed to UV and held in a minimal medium before plating onto rich or minimal agar plates. It was found that, under these conditions, holding cells in minimal (nongrowth) conditions resulted in mutations that enabled cells to grow on minimal media. Using this observation as a starting point, a mutant was isolated that failed to mutate to auxotrophy under the prescribed conditions, and the gene responsible for this phenomenon (mutation frequency decline) was named mfd. Later work revealed that mfd encoded a translocase that recognizes a stalled RNA polymerase (RNAP) at damage sites and binds to the stalled RNAP, recruits the nucleotide excision repair damage recognition complex UvrA2UvrB to the site, and facilitates damage recognition and repair while dissociating the stalled RNAP from the DNA along with the truncated RNA. Recent single-molecule and genome-wide repair studies have revealed time-resolved features and structural aspects of this transcription-coupled repair (TCR) phenomenon. Interestingly, recent work has shown that in certain bacterial species, mfd also plays roles in recombination, bacterial virulence, and the development of drug resistance.