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:The unique nucleolar environment, the repetitive nature of ribosomal DNA (rDNA), and especially the possible involvement of RNA polymerase I (RNAPI) in transcription-coupled repair (TCR) have made the study of repair of rDNA both interesting and challenging. TCR, the transcription-dependent, preferential excision repair of the template strand compared with the nontranscribed (coding) strand has been clearly demonstrated in genes transcribed by RNAPII. Whether TCR occurs in rDNA is unresolved. In the present work, we have applied analytical methods to map repair events in rDNA using data generated by the newly developed XR-seq procedure, which measures excision repair genome-wide with single-nucleotide resolution. We find that in human and mouse cell lines, rDNA is not subject to TCR of damage caused by UV or by cisplatin.
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 developed a method for genome-wide mapping of DNA excision repair named XR-seq (excision repair sequencing). Human nucleotide excision repair generates two incisions surrounding the site of damage, creating an ∼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 bidirectional enhancer RNA (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.
Project description:Gafchromic film has been used for X-ray dose measurement in diagnostic examinations. Their use has been initiated for three-dimensional X-ray dose measurement by using the high-resolution characteristics of Gafchromic films in computed tomography. However, it is necessary to solve the problem of nonuniform thickness in the active layers of Gafchromic films. A double exposure technique using X-rays is performed in therapeutic radiology; it is difficult to use in a diagnostic examination because of the heel effect. Therefore, it is suggested that ultraviolet (UV) rays be substituted for X-rays. However, the appropriate UV wavelength is unknown. In this study, we aimed to determine which UV wavelengths are effective to expose Gafchromic XR-RV3 and XR-SP2. UV lamps with peak wavelengths of 245 nm, 310 nm, and 365 nm were used. The three UV wavelengths were used to irradiate Gafchromic XR-RV3 and XR-SP2 films for 60 min, and irradiation was repeated every 60 min for 600 min thereafter. Films were scanned after each irradiation period on a flatbed scanner. The images were split into their red-green-blue components, and red images were stored using ImageJ version 1.44o image analysis software. Regions of interest (ROI), 0.5 inches in diameter, were placed at the centers of the subtracted Gafchromic film images, and graphs of UV irradiation duration and mean pixel values were plotted. There were reactions to UV-A on both Gafchromic XR-RV3 and XR-SP2; those to UV-B were moderate. However, UV-C demonstrated few reactions with Gafchromic XR-RV3 and XR-SP2. From these results, irradiation with UV-A may be able to correct nonuniformity errors. Uniform UV-A irradiation of Gafchromic films with large areas is possible, and UV rays can be used as a substitute for X-rays in the double exposure technique.
Project description:We recently developed a high-resolution genome-wide assay for mapping DNA excision repair named eXcision Repair-sequencing (XR-seq) and have now used XR-seq to determine which regions of the genome are subject to repair very soon after UV exposure and which regions are repaired later. Over a time course, we measured repair of the UV-induced damage of cyclobutane pyrimidine dimers (CPDs) (at 1, 4, 8, 16, 24, and 48 h) and (6-4)pyrimidine-pyrimidone photoproducts [(6-4)PPs] (at 5 and 20 min and 1, 2, and 4 h) in normal human skin fibroblasts. Each type of damage has distinct repair kinetics. The (6-4)PPs are detected as early as 5 min after UV treatment, with the bulk of repair completed by 4 h. Repair of CPDs, which we previously showed is intimately coupled to transcription, is slower and in certain regions persists even 2 d after UV irradiation. We compared our results to the Encyclopedia of DNA Elements data regarding histone modifications, chromatin state, and transcription. For both damage types, and for both transcription-coupled and general excision repair, the earliest repair occurred preferentially in active and open chromatin states. Conversely, repair in regions classified as "heterochromatic" and "repressed" was relatively low at early time points, with repair persisting into the late time points. Damage that remains during DNA replication increases the risk for mutagenesis. Indeed, late-repaired regions are associated with a higher level of cancer-linked mutations. In summary, we show that XR-seq is a powerful approach for studying relationships among chromatin state, DNA repair, genome stability, mutagenesis, and carcinogenesis.
Project description:Subunit 2 of DNA damage-binding protein complex (DDB2) is an early sensor of nucleotide excision repair (NER) pathway for eliminating DNA damage induced by UV radiation (UVR) and cisplatin treatments of mammalian cells. DDB2 is modified by ubiquitin and poly(ADP-ribose) (PAR) in response to UVR, and these modifications play a crucial role in regulating NER. Here, using immuno-analysis of irradiated cell extracts, we have identified multiple post-irradiation modifications of DDB2 protein. Interestingly, although the DNA lesions induced by both UVR and cisplatin are corrected by NER, only the UV irradiation, but not the cisplatin treatment, induces any discernable DDB2 modifications. We, for the first time, show that the appearance of UVR-induced DDB2 modifications depend on the binding of DDB2 to the damaged chromatin and the participation of functionally active 26S proteasome. The in vitro and in vivo analysis revealed that SUMO-1 conjugations comprise a significant portion of these UVR-induced DDB2 modifications. Mapping of SUMO-modified sites demonstrated that UVR-induced SUMOylation occurs on Lys-309 residue of DDB2 protein. Mutation of Lys-309 to Arg-309 diminished the DDB2 SUMOylation observable both in vitro and in vivo. Moreover, K309R mutated DDB2 lost its function of recruiting XPC to the DNA damage sites, as well as the ability to repair cyclobutane pyrimidine dimers following cellular UV irradiation. Taken together, our results indicate that DDB2 is modified by SUMOylation upon UV irradiation, and this post-translational modification plays an important role in the initial recognition and processing of UVR-induced DNA damage occurring within the context of chromatin.
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: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 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- to 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 at early time points followed by predominantly nontranscribed strand repair at later stages. We have also characterized the excision repair level as a function of the transcription level. The availability of high-resolution and dynamic repair maps should aid in future repair and mutagenesis studies in this model organism.