Single Nucleotide Resolution Analysis of Nucleotide Excision Repair of Ribosomal DNA in Humans and Mice
ABSTRACT: In the present work we have applied analytical methods to map repair events in rDNA using data generated by the newly developed XR-seq genome-wide single nucleotide repair technology. We find that in human and mouse cell lines, rDNA is not subject to TCR of damage caused by UV or by cisplatin. Overall design: We perform XR-seq in mouse skin fibroblast under UV irradiation and collect cells after incubation 3 hours. For human cell lines NHF1, CSB and XPC, we perform XR-seq under UV irradiation and collect cells after incubation 1 hour. For GM12878, we perform XR-seq under cisplatin and collect cells after incubation 2 hours. Then we mapped all the reads to rDNA or DHFR. This dataset includes re-analysis of five GSE67941 Samples and two GSE82213 Samples.
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: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 tested the impact of clock time on excision repair of cisplatin-induced DNA damage at single nucleotide resolution across the genome in mouse liver and kidney. We found that genome repair is controlled by two circadian programs. Overall design: we perform XR-seq in mouse kidney and liver at different Zeitgeber time. After we injected the mouse with cisplatin 10mg / kg 2 hours, we sacrificed the mouse at indicated time and took the kidneys and liver. then lysed tissue and did TFIIH IP and cisplatin IP for construct the XR-seq library and sequence.
Project description:The fate of RNAPII in the course of the transcription-coupled repair pathway is unclear. To address this problem we have used methods to control transcription, so as to initiate a discrete ‘wave’ of elongation complexes. We also used methods to identify where elongation complexes and transcription-repair coupling events are located in genes throughout the genome. Overall design: XP-C cells which is deficient in the global repair were treated with different DRB regimens. After UV irradiation, XR-seq and mNET were performed to analyze the repair and the localization of elongating RNA polymerase II
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: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: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:Alternative splicing (AS) is the main process that amplifies DNA information and is a crucial target of signaling cascades resulting from UV irradiation of human cells. The specific impact of UV-induced cyclobutane pyrimidine dimers (CPDs) at the transcriptional and AS levels has not been addressed. By using a CPD photolyase, a flavoenzyme that directly reverts CPDs, we analyze the contribution of this lesion to the expression program in human keratinocytes. Overall design: mRNA profiles of human keratinocytes (HaCaT cell line) expressing the CPD photolyase from Potorous tridactylus in three different conditions: 1. Control: Non treated cells and inactive photolyase. 2. UV treated cells: 15 J/m2 (UVC, 254 nm) and inactive photolyase, harvested 6 hours after UV irradiation and 3. Same as before but with an active photolyase due to exposure to white light for 2 hours.
Project description:We developed a novel approach, m6A-seq, for high-resolution mapping of the transcriptome-wide m6A landscape, based on antibody-mediated capture followed by massively parallel sequencing. Identification of m6A modified sequences in HepG2 cells. HepG2 cells were incubated with either IFNg (200ng/ml) or HGF/SF (10 ng/ml) over night. Stress effects were tested in HepG2 cells by either 30 minutes incubation at 43ºC (heat shock) or UV irradiation of 0.04 J/cm2 followed by 4 hours of recovery in normal growing conditions prior to harvesting using Trypsin.
Project description:Genome-wide mapping of transcription factor binding is generally performed by chemical protein-DNA crosslinking, followed by chromatin immunoprecipitation and deep sequencing (ChIP-seq). Here we present the ChIP-seq technique based on photochemical crosslinking of protein-DNA interactions by high-intensity ultraviolet (UV) laser irradiation in living mammalian cells (UV-ChIP-seq). UV laser irradiation induces efficient and instant formation of covalent “zero-length” crosslinks exclusively between nucleic acids and proteins that are in immediate contact, thus resulting in a “snapshot” of direct protein-DNA interactions in their natural environment. We applied UV-ChIP-seq for genome-wide profiling of the sequence-specific transcriptional repressor B-cell lymphoma 6 (BCL6) in human diffuse large B-cell lymphoma (DLBCL) cells. Our approach resulted in sensitive and precise protein-DNA binding profiles, highly enriched in canonical BCL6 DNA sequence motifs. UV-ChIP-seq also revealed numerous previously undetectable BCL6 binding sites, particularly in more condensed, inaccessible areas of chromatin. Overall design: Genome-wide mapping of BCL6-DNA interactions by replicate UV-ChIP-seq and corresponding control experiments. To control for enrichment of non-crosslinked protein-DNA interactions we performed ChIP-seq using non-irradiated cells (-UV control ChIP). Furthermore, unspecific antibody binding and input loading was controlled by sequencing of IgG enriched DNA fragments (UV IgG control) and input DNA fragments (UV input DNA) following UV irradiation, respectively. To compare photochemical (UV) with conventional chemical (FA) crosslinking, we performed BCL6 FA ChIP-seq and corresponding control experiments (FA input DNA, FA IgG control).