Journal: Nature structural & molecular biology

Article Title: Damage sensor role of UV-DDB during base excision repair

doi: 10.1038/s41594-019-0261-7

Figure Lengend Snippet: (a). Binding isotherms for THF, CPD, 8-oxoG:C, 8-oxoG:A and undamaged DNA. Data is plotted as mean ± s.e.m. of three independent experiments. (b). Schematic representation of the DNA substrate containing 8-oxoG and the proposed reaction scheme. (c) . Stimulation of OGG1 incision kinetics by UV-DDB. OGG1 (50nM) was incubated with dsDNA (50nM) containing 8-oxoG:C in the absence (−) or presence (+) of UV-DDB (16nM) at 37°C. Aliquots were withdrawn at each time point and analyzed on a 10% denaturing polyacrylamide gel. Positions of the un-cleaved full-length substrate and incised product are indicated by arrows. (d). Quantification of the stimulation of OGG1 incision kinetics by UV-DDB in (c). Incision product formation was quantified using GelAnalyzer software. The incision percentage was plotted as mean ± s.d. from seven independent experiments, each run on duplicate gels. (e). Schematic representation of the DNA substrate containing a THF-site and the proposed reaction scheme. (f). Stimulation of APE1 incision kinetics by UV-DDB. APE1 (0.5nM) was incubated with the THF37 dsDNA (50nM) in the absence (−) or presence (+) of UV-DDB (10nM) at room temperature. Aliquots were withdrawn at each time point and analyzed on a 10% denaturing polyacrylamide gel. Positions of the un-cleaved full-length substrate and incised product are indicated by arrows. (g). Quantification of the stimulation of APE1 incision kinetics by UV-DDB in (f). Quantification of the incision product formation was performed using GelAnalyzer software. The incision percentage was plotted as mean ± s.d. from three independent experiments, each run on duplicate gels.

Article Snippet: Recombinant human OGG1 protein was purchased from Novus Biologicals or purified from bacterial cells as a GST fusion as previously described .

Techniques: Binding Assay, Incubation, Software

Journal: Nature structural & molecular biology

Article Title: Damage sensor role of UV-DDB during base excision repair

doi: 10.1038/s41594-019-0261-7

Figure Lengend Snippet: (a). Experimental design of DNA tightrope assay to study UV-DDB facilitated dissociation of OGG1 or APE1. (Top) Long DNA substrates with defined abasic sites (THF) every 2 kb are suspended between silica beads. (Bottom, left) His-tagged OGG1 or APE1 are labeled with primary mouse-anti-His antibody and secondary goat-anti-mouse antibody conjugated to a 605 nm Qdot. (Bottom, right) Flag-tagged UV-DDB is bound by primary goat-anti-Flag antibody. (b). Motility of 605Qdot-labeled OGG1 (green: motile, gray: non-motile) on DNA tightropes containing abasic sites (THF) in the absence or presence of 1X (2.6nM) or 10X (26nM) UV-DDB. (c). Motility of 605Qdot-labeled APE1 (green: motile, gray: non-motile) on DNA tightropes containing abasic sites (THF) in the absence or presence of 1X or 10X UV-DDB. (d). Comparison of dissociation percentage of 605Qdot-labeled OGG1 in the absence or presence of unlabeled 1X or 10X UV-DDB. Bar graph data shown as weighted means ± weighted SDs with three independent experiments. (** p< 0.01; *** p<0.001 by two-tailed Student’s t test). (e). Comparison of dissociation percentage of 605Qdot-labeled APE1 in the absence or presence of unlabeled 1X or 10X UV-DDB. Bar graph data are represented as weighted means ± weighted SDs with three independent experiments. (** p< 0.01; *** p<0.001 by two-tailed Student’s t test). (f). Effects of UV-DDB on the life times of OGG1-DNA complexes. Data plotted as the mean ± SEM from three independent experiments. For each condition, survival fraction decay is fit to a single exponential decay function to obtain the half-life. (g). Effects of UV-DDB on the life times of APE1-DNA complexes. Data plotted as the mean ± SEM from three independent experiments. For each condition (without UV-DDB or with 1X/10X UV-DDB), survival fraction decay is fit to a single exponential decay function to obtain the half-life.

Article Snippet: Recombinant human OGG1 protein was purchased from Novus Biologicals or purified from bacterial cells as a GST fusion as previously described .

Techniques: Labeling, Comparison, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Damage sensor role of UV-DDB during base excision repair

doi: 10.1038/s41594-019-0261-7

Figure Lengend Snippet: (a). Schematic of the DNA tightrope assay. Long DNA substrates with abasic sites every 2 kb were suspended between 5μm poly-L-lysine coated silica beads. Anti-His primary antibody was used to link the His-tagged OGG1 or APE1 to the 605Qdot. Biotin conjugated anti-Flag primary antibody was used to link Flag-tagged UV-DDB to streptavidin-coated 705Qdot. Uniquely labeled OGG1/APE1 and UV-DDB were observed interacting on abasic DNA tightropes in real time and their behavior and frequency of co-localization was recorded. (b) . Venn diagram showing number of proteins that co-localized (yellow) on abasic (THF) tightropes or were observed separately for 605Qdot-labeled OGG1 (green) with 705Qdot-labeled UV-DDB (red) in the dual-color assay. (c). Image of co-localized (yellow) Qdot-labeled OGG1 (green) and UV-DDB (red) on abasic (THF) tightrope suspended between beads. Scale bar represents 2.5μm. Arrow points to co-localized particle. (d). Kymograph of co-localized OGG1 and UV-DDB. Top, OGG1 (green); middle, UV-DDB (red); bottom, merged (yellow). Horizontal and vertical scale bars represent 50s and 2kb, respectively. (e). Venn diagram showing number of proteins that co-localized (yellow) on abasic (THF) tightropes or were observed separately for 605Qdot-labeled APE1 (green) with 705Qdot-labeled UV-DDB (red) in the dual-color assay. (f). Image of co-localized (yellow) Qdot-labeled APE1 (green) and UV-DDB (red) on abasic (THF) tightrope suspended between beads. Scale bar represents 2.5μm. Arrow points to co-localized particle. (g). Kymograph of co-localized APE1 and UV-DDB. Top, APE1 (green); middle, UV-DDB (red); bottom, merged (yellow). Horizontal and vertical scale bars represent 50s and 2kb, respectively. See also and and .

Article Snippet: Recombinant human OGG1 protein was purchased from Novus Biologicals or purified from bacterial cells as a GST fusion as previously described .

Techniques: Labeling

Journal: Nature structural & molecular biology

Article Title: Damage sensor role of UV-DDB during base excision repair

doi: 10.1038/s41594-019-0261-7

Figure Lengend Snippet: (a). Schematic representation of the in vitro BER substrate and the expected BER products after gap-filling (insertion of [α 32 P]-dCTP) and ligation steps. (b). BER time course and the influence of UV-DDB on the activity of BER components. UDG pretreated DNA (200 nM) was incubated with APE1 (1 nM) in the absence (−) or presence (+) of UV-DDB (50 nM), Pol β (20 nM) and DNA ligase III (250 nM). The migration positions of the ligated BER product and unligated BER intermediate are indicated. The results shown are representative of three independent experiments. Quantitation of lane 1 versus lane 9, from three independent experiments shows a 30-fold difference in [α 32 P]-dCTP incorporation stimulated by UV-DDB. (c). Cell survival curves of BJ-hTERT cells transfected with scrambled or DDB2 siRNA and treated with a range of concentration of KBrO 3 . Data represent mean ± SEM from three independent experiments, each performed in triplicate. (* p<0.05; ** p<0.01 by two-tailed Student’s t test) (d). Schematic description of the TRF1 FAP system. U2OS cells stably expressing the TRF1 FAP were treated with 100nM dye (MG2I) and light (660nm) for 10 minutes to generate singlet oxygen at the telomeres leading to the formation of 8-oxoG. (e). Immunofluorescence images depicting the recruitment of mCherry-DDB2 to telomeres after specifically inducing 8-oxoG at the telomeric region. Scale bar: 5μm (f). Bar graph showing quantification of (e), where horizontal bars indicate mean ± SEM from two independent experiments (*** p<0.001 by one-way ANOVA). (g). Bar graph quantifying the DDB2 and OGG1 foci at telomeres over a 3-hour time course. Data represents mean ± SEM from two independent immunofluorescence experiments. (*p<0.05; ** p<0.01 by two-way ANOVA).

Article Snippet: Recombinant human OGG1 protein was purchased from Novus Biologicals or purified from bacterial cells as a GST fusion as previously described .

Techniques: In Vitro, Ligation, Activity Assay, Incubation, Migration, Quantitation Assay, Transfection, Concentration Assay, Two Tailed Test, Stable Transfection, Expressing, Immunofluorescence

Journal: Nature structural & molecular biology

Article Title: Damage sensor role of UV-DDB during base excision repair

doi: 10.1038/s41594-019-0261-7

Figure Lengend Snippet: Schematic representation of the proposed BER pathway including UV-DDB is illustrated. UV-DDB appears to be rapidly recruited to damaged sites in chromatin and help facilitate processing by OGG1, MUTYH (not shown) and APE1. Biochemical and single molecule data suggest that UV-DDB transiently associates with OGG1 or APE1 at abasic sites to increase their turnover and stimulated BER (see text for description).

Article Snippet: Recombinant human OGG1 protein was purchased from Novus Biologicals or purified from bacterial cells as a GST fusion as previously described .

Techniques:

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A) OGG1 binds RNA containing 8-oxoGua. The secondary structure of the undenatured single-strand probe was predicted using software as described in Materials and Methods. The probe contains a single 8-oxoGua (marked in red) at the end of GGGG. EMSA was performed using single-stranded RNA (8oxo-rG), either denatured at 95°C for 5 min (Lanes 1–4) or undenatured (Lanes 5–8). Additionally, EMSA was conducted with the double-stranded RNA probe (8oxo-rG: rC), where 8oxo-rG anneals with its complementary genome sequence (rC) (Lanes 9–12). (B) Representative EMSA image demonstrating OGG1’s binding affinity for annealed RNA containing 8-oxoGua. (C) Representative image showing OGG1’s excision activity on 8-oxoGua within DNA, contrasting with its inactivity towards RNA. (D) EMSA visualization indicating that TH5487 impedes OGG1’s binding to RNA harboring 8-oxoGua. (E) EMSA depiction showing OGG1’s interaction with a DNA-RNA hybrid that includes the gene-start (GS) motif. Total RNA (1 μg) extracted from virocells (MOI = 1, 24 hpi) was hybridized with Cy5-labelled DNA probes (20 nM) containing the intergenic region (IG), the gene-start (GS), and the gene-end (GE) of G gene. Following incubation with recombinant OGG1 (100 nM), the assay proceeded to EMSA. Oligo sequences for EMSA and OGG1 glycosylase activity are listed in . n = 3 biological replicates.

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques: Software, Sequencing, Binding Assay, Activity Assay, Incubation, Recombinant

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A) Left panel: Strategy for enrichment of viral RNAs interacting with OGG1 and N protein from virocells after formaldehyde crosslinking. Treatment with TH5487 (10 μM) was initiated post inoculum (MOI = 1, 24 hpi). RNA-immunoprecipitation (RIP) was carried out using antibodies against OGG1 or RSV N protein. Right panel: Quantification of RSV genomic RNA levels after antibody pull-down, determined by qRT-PCR. Fold change was calculated by 2^- [Ct (test Ab)-Ct (negative Ab)]. n = 3 biological replicates. Statistical analysis was conducted using an unpaired Student’s t test, with results shown as means ± SD. *** p <0.001. (B) OGG1 RIP-Seq analysis in virocells (MOI = 1, 24 hpi) illustrating the distribution of RSV-specific reads across two independent experiments (visualized in blue and red tracks). Data are presented as the log2 ratio of reads obtained from anti-OGG1 immunoprecipitation relative to input RNA, mapped against the RSV genome (GenBank: M74568.1). Peaks were called positive if the log2 enrichment score was ≥1. (C) Annotation and categorization of cellular RNA reads obtained from OGG1 RIP-Seq aligned to the human genome HG38 (GenBank: GCA_000001405.29), presented as the percentage of OGG1-RIP peaks. UTR, untranslated region of mRNAs; CDS, exon regions that code for protein; und., undefined. Fig 4A created with Biorender.com .

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Immunoprecipitation

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A) LC_MS/MS identification of RSV proteins co-immunoprecipitated with OGG1 (RSV MOI = 1, 24 hpi). The analysis was conducted on complexes isolated using an anti-OGG1 antibody (for endogenous OGG1) and anti-FLAG antibody (for transgenically expressed OGG1). Proteins are listed according to the number of significant peptides identified. ND not detected. (B) The entire amino acid sequence of the RSV N protein was visualized, with residues identified by LC_MS/MS highlighted in red. (C) The peptides identified by LC_MS/MS in red were visualized within the three-dimensional structure of the N protein, with RNA depicted in green. NTD, N terminal domain. CTD, C terminal domain.

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Isolation, Sequencing

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A) Immunoblot (IB) analysis of co-immunoprecipitated extracts from Flag-OGG1 expressing hSAECs infected with RSV (MOI = 1, 24 hpi). Co-immunoprecipitation was performed using an anti-FLAG antibody or control IgG, and the blots were subsequently probed with an anti-RSV antibody. Anti-Actin immunoblotting from whole cell lysates served as a loading control. An arrow indicates the N protein. (B) Immuno-fluorescence staining of OGG1 and RSV N protein. hSAECs were mock or RSV-infected (MOI = 1), and cells were fixed at 18 and 24 hpi. The co-localization measurement between fluorophores (Alexa 594 vs. Alexa 488) was assessed using the intensity of individual fluorophore pixels, calculated as the Pearson correlation coefficient (R). Scale bars: 20 μm. (C) Proximity ligation assay (PLA) shows OGG1 and N interactions (MOI = 1, 24 hpi). PLA signals were absent when control IgG were applied. Scale bars, 50 μm. (D-E) GST pull-down assays demonstrating the interaction between OGG1 and the N protein. Immobilized GST-tagged OGG1 (100 nM) was incubated with increasing concentrations of His-tagged N, followed by washing, SDS-PAGE and immunoblotting using an anti-N antibody. (D) In a reciprocal setup, His-tagged N protein (100 nM) was incubated with varying concentrations of GST-OGG1, followed by immunoblotting with an anti-OGG1 antibody (E).

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques: Western Blot, Immunoprecipitation, Expressing, Infection, Control, Fluorescence, Staining, Proximity Ligation Assay, Incubation, SDS Page

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: (A-B) hSAECs were infected with RSV (MOI = 0.1) and viral titers were assessed 3 days post-infection in scenarios where OGG1 expression was either (A) down-regulated via siRNA or (B) completely eliminated by CRISPR/Cas9 knockout. NT, non-targeting siRNA. The knockout was overcompensated by ectopic expression of OGG1. The lower panel displays immunoblot analysis of OGG1 expression in cell lysates, with Actin serving as a loading control. (C) Following 1-hour post inoculation (MOI = 0.1), hSAECs were treated with TH5487, TH2840, or O8, and viral titers were determined by plaque assays (3 dpi). In D-I, hSAECs were infected with RSV (MOI = 0.5), and total RNA was extracted first at indicated times post-infection. Experiments include OGG1 knockout by CRISPR/Cas9 in hSAECs (D and G), down regulation of OGG1 expression by siRNA (E and H), and inhibition of OGG1’s reading function by TH5487 (F and I). (D-F) mRNA was isolated using Oligo dT beads, and G mRNA level was quantified by RT-qPCR. (G-I) After mRNA isolation, RSV gRNA levels were determined via RT-qPCR. n = 3. Statistical analysis was conducted using an unpaired Student’s t test, with results shown as means ± SD. Significance levels are indicated as * p <0.05, ** p < 0.01, *** p < 0.001. (J) Efficiency of primer extension on RNA template containing 8-oxoGua. Moloney murine leukemia virus reverse transcriptase (RT) was used in the assay. H 2 O 2 , 800 μM. OGG1, 50 nM. P indicates primer. E indicates extension.

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques: Infection, Expressing, CRISPR, Knock-Out, Western Blot, Control, Inhibition, Isolation, Quantitative RT-PCR, Virus, Reverse Transcription

Journal: PLOS Pathogens

Article Title: 8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus

doi: 10.1371/journal.ppat.1012616

Figure Lengend Snippet: Elevated ROS levels strategically alter host mechanisms to detect oxidative modifications in viral RNA. (A) OGG1 assumes a pivotal role in this process, co-opted by viral nucleoproteins to facilitate precise base pairing in progeny genomes. (B) Inhibiting OGG1’s ability to detect 8-oxoGua compromises the accuracy of viral replication, resulting in reduced viral progeny production. RdRp, RNA-dependent RNA polymerase. Figure created with Biorender.com .

Article Snippet: Specifically, GST-tagged OGG1 (TP720109, OriGene, Rockville, MD) was immobilized onto Glutathione Agarose, and subsequently incubated with varying concentrations of His-tagged nucleoprotein (NP-424V, Creative BioMart, Shirley, NY), or His-tagged M2-1 protein (purified locally).

Techniques: