Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis
ABSTRACT: We recently developed a high-resolution genome wide assay for mapping DNA excision repair named eXcision Repair-sequencing (XR-seq) (GEO accession: GSE67941) We have now used this assay to assay the effect of chromatin state on DNA repair. Here we report the results of a time-course of the repair of the UV induced damages cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs] in normal human skin fibroblasts. Comparison of this data to histone modification and DNA-seq maps (ENCODE) revealed initial repair of both damages is enriched in open and active chromatin states, whereas repair in heterochromatic and repressed chromatin states is relatively low and persists to later time points. We performed XR-seq for two types of UV induced damages (CPD and (6-4)PP) at multiple time points after UV irradiation, in normal NHF1, and CS-B (CS1ANps3g2, GM16095) fibroblast cell lines. Two biological replicates were performed for each experiment in which independent independent cell populations were UV treated and subjected to XR-seq. For assaying CPD repair, cells were irradiated with 10J/m2 and for assaying (6-4)PP cells were irradiated with 20J/m2. Raw data for the 1h time points of (6-4)PP repair are the same as in GEO accession GSE67941).
Project description:We developed a method for genome-wide mapping of DNA excision repair named XR-seq (eXcision Repair-seq). Human nucleotide excision repair generates two incisions surrounding the site of damage, creating a ~30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of repair of two UV-induced DNA damages in human cells, cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts ((6-4)PPs). In wild-type cells, CPD repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled excision repair or general excision repair isolated the contribution of each pathway to the overall repair pattern, and showed that transcription-coupled repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled repair at sites of divergent gene promoters and bi-directional eRNA production at enhancers. XR-seq data also uncovered the repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA repair in human cells. We have performed XR-seq for two types of UV-induced damages (CPD and (6-4)PP) in three different cell lines: NHF1, XP-C (XP4PA-SV-EB, GM15983)), and CS-B (CS1ANps3g2, GM16095). Two biological replicates were performed for each experiment, in which independent cell populations were UV treated and subjected to XR-seq.
Project description:UV-induced DNA lesions are an important contributor to mutagenesis and cancer, but it is not fully understood how the chromosomal landscape influences UV lesion formation and repair. We have used a novel high-throughput sequencing method to precisely map UV-induced cyclobutane pyrimidine dimers (CPDs) at nucleotide resolution throughout the yeast genome. Analysis of CPD formation reveals that nucleosomal DNA having an inward rotational setting is protected from CPD lesions. In strongly positioned nucleosomes, this nucleosome 'photofootprint' overrides intrinsic dipyrimidine sequence preferences for CPD formation. CPD formation is also inhibited by DNA-bound transcription factors, in effect protecting important DNA elements from UV damage. Analysis of CPD repair revealed a clear signature of efficient transcription-coupled nucleotide excision repair. Repair was less efficient at translational positions near a nucleosome dyad and at heterochromatic regions in the yeast genome. These findings define the roles of nucleosomes and transcription factors in UV damage formation and repair. UV mapping data was analyzed for yeast cells irradiated with 125J/m2 and allowed to repair for 0hr (2 samples), 20 minutes, 1 hour, or 2 hours. Data is also included for naked DNA irradiated with UV 60 or 90 J/m2
Project description:Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, is the major cause of lung cancer. It forms covalent DNA adducts after metabolic activation and induces mutations. We have developed a method capturing oligonucleotides carrying bulky base adducts, including UV-induced cyclobutane pyrimidine dimers (CPDs) and BaP diol epoxide-deoxyguanosine (BPDE-dG), which are removed from the genome by nucleotide excision repair. The isolated oligonucleotides are ligated to adaptors and after damage-specific immunoprecipitation the adaptor-ligated oligonucleotides are converted to dsDNA with an appropriate translesion DNA synthesis (TLS) polymerase followed by PCR amplification and next-generation sequencing (NGS) to generate genome-wide repair maps. This method, which we have named translesion eXcision Repair-sequencing (tXR-seq) does not depend on repair/removal of the damage in the excised oligonucleotides and hence it is applicable to essentially all DNA damages processed by nucleotide excision repair. Here, we present the excision repair maps for CPDs and BPDE-dG adducts generated by tXR-Seq for the human genome. Additionally, we observe novel sequence specificity of BPDE-dG excision repair by using tXR-seq. Overall design: We performed tXR-seq for UV-induced CPD and BPDE-dG repair in GM12878 cell line. We also compared our tXR-seq data with our published dataset from XR-seq for CPD and (6-4)PP (GEO accession: GSE67941).
Project description:The rates at which lesions are removed by DNA repair can vary widely throughout the genome with important implications for genomic stability. We measured the distribution of nucleotide excision repair (NER) rates for UV induced lesions throughout the yeast genome. By plotting these repair rates in relation to all ORFs and their associated flanking sequences, we reveal that in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organised within the genome. We compared genome-wide DNA repair rates in wild type and in RAD16 deleted cells, which are defective in the global genome-NER (GG-NER) sub-pathway, demonstrating how this alters the normal distribution of NER rates throughout the genome. We examine the genomic locations of global genome NER factor binding in chromatin before and after UV irradiation, and reveal that GG-NER is organized and initiated from specific locations. By controlling the chromatin occupancy of the histone acetyl transferase Gcn5, the GG-NER complex regulates the histone H3 acetylation status and chromatin structure in the vicinity of these genomic sites to promote the efficient DNA repair of UV induced lesions. This demonstrates that chromatin remodeling during the GG-NER process is organized into domains in the genome. Importantly, we demonstrate that deleting the histone modifier GCN5, an accessory factor required for chromatin remodeling during GG-NER, significantly alters the genomic distribution of NER rates. These observations could have important implications for the effect of histone and chromatin modifiers on the distribution of genomic mutations acquired throughout the genome.
Project description:UV-induced DNA damage is the major carcinogen of skin cancer. We developed HS-Damage-seq to map UV damage with unprecedented sensitivity and resolution in human cells. While the distribution of UV-induced damage are mainly determined by sequence context and essentially uniform throughout the genome, transcription factors can affect damage formation at specific positions. With time-course experiments, we observed changes in damage distribution at particular genomic loci and chromatin states, which reflects the nucleotide excision repair preferences and complements the repair maps by XR-seq. Our results demonstrated that while initial damage is uniformly distributed, they are heterogeneously repaired throughout the genome. The combination of damage and repair maps provides a holistic perspective of UV damage and repair in the human genome. Overall design: 38 samples were analyzed for (6-4)PP and CPD. Two main experiments were performed: time-course (with NHF1 cell line) and cell vs naked DNA (with GM12878 cell line). Each sample has a biological replicate. We also have control samples which are the undamaged samples and reference genome samples for two cell lines.
Project description:We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs] in the Saccharomyces cerevisiae genome. We find that these photoproducts are removed from the genome primarily by incisions 13-18 nucleotides 5’ and 6-7 nucleotides 3’ to the UV damage that generate 21-27 nt-long excision products. Analyses of the excision repair kinetics both in single genes and at the genome-wide level reveal strong transcription-coupled repair of the transcribed strand (TS) at early time points followed by predominantly non-transcribed strand (NTS) repair at later stages. We have also characterized the excision repair level as a function of transcription level. The availability of high-resolution and dynamic repair maps should aid in future repair and mutagenesis studies in this model organism. Overall design: We performed XR-seq for UV-induced CPD and (6-4)PP repair in wild type yeast cells. Our analysis intergreted the published datasets from RNA-seq and NET-seq (GEO accession: GSE68484) and CPD-seq (GEO accession: GSE79977).
Project description:Paralytic peptide (PP) participates in diverse physiological processions as an insect cytokine, such as immunity controls, paralysis induction, regulation of cell morphology and proliferation. To investigate the molecular mechanism underlying those physiological activities, we systematically investigated the global phosphorylation events in fat body of silkworm larvae induced by PP through label-free quantitative phosphoproteomics.
Project description:Platinum chemotherapies induce damages in DNA that distort the helical structure. In human cells, these adducts are removed primarily by the Nucleotide Excision Repair pathway. In this study, we mapped both cisplatin and oxaliplatin induced damages and their repair at single nucleotide resolution across the human genome. Overall design: We performed Damage-seq to map 2 types of damages in two different human cell lines. The damages were cisplatin and oxaliplatin induced DNA adducts. The two cell lines were normal human skin fibroblasts NHF1 and the GM12878 human lymphoblastoid cells. As control, cisplatin damages were induced in genomic DNA from GM12878. For comparison to repair, XR-seq was performed for cisplatin and oxaliplatin treated GM12878 cells.
Project description:Similar to other plant-parasitic nematodes, root lesion nematodes possess an array of enzymes that are involved in degradation of the plant cell wall. Here we report the identification of a gene encoding a cell wall degrading enzyme, pectin methylesterase PME (EC 188.8.131.52), in the root lesion nematode Pratylenchus penetrans. Both genomic and coding sequences of the gene were cloned for this species, showing the presence of four introns that excluded a potential bacterial contamination. Expression of the Pp-pme gene was localized in the esophageal glands of P. penetrans as determined by in situ hybridization. Temporal expression of Pp-pme in planta was validated for early time points of infection. The possible function and activity of the gene were assessed by transient expression of Pp-pme in N. benthamiana plants via a Potato virus X-based vector. To our knowledge, this is the first report on identification and characterization of a PME gene within the phylum Nematoda.