Journal: Nature Communications

Article Title: Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins

doi: 10.1038/s41467-022-28642-9

Figure Lengend Snippet: a , c Representative images showing recruitment of GFP-XPC ( a ) or GFP-XPA ( c ) to 8-oxoG at telomeres after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment in U2OS WT and DDB2 KO cells, 30 min post treatment. b , d Percentage telomeres colocalized with GFP-XPC ( b ) or GFP-XPA ( d ) after treatment, over a period of 3 h. e , g Representative images of GFP-XPC ( e ) or GFP-XPA ( g ) accumulation at damaged telomeres 30 min after dye plus light treatment in cells pretreated with transcription inhibitors α-amanitin and THZ1. f , h Quantification of e ( f ) and g ( h ). i , j Colocalization of GFP-XPC with telomeres after dye plus light treatment in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. k , l Colocalization of GFP-XPA with telomeres after dye plus light treatment in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. Data ( a – l ) represents mean ± SEM from two independent experiments. “ n ” represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test) ( b , d , f , h ) and Student’s two-tailed t -test ( j , l ): ** p < 0.01; *** p < 0.001; **** p < 0.0001. Scale: 5 µm. Source data are provided as a Source Data file. (See also Supplementary Fig. ).

Article Snippet: Primary antibodies used: DDB2 (1:1000; abcam #ab181136), OGG1(1:1000; abcam #124741), Cul4A (1:1000; CST #2699 S), DDB1 (1:1000; Invitrogen #37-6200), XPC (1:1000; CST #12701 S), CSB (1:1000; abcam #ab96089), mCherry (1:1000; Abcam #ab167453), β-actin (1:30,000; Sigma #A2228).

Techniques: Transfection, Control, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins

doi: 10.1038/s41467-022-28642-9

Figure Lengend Snippet: Treatment of cells expressing FAP-TRF1 with dye (100 nM, 15 min) plus light (660 nm, 10 min) introduces 8-oxoG lesions at telomeres. In the DDB2-dependent repair pathway, DDB2 recognizes 8-oxoG lesions and facilitates chromatin relaxation through chromatin decompaction allowing the recruitment of XPC and OGG1 to the damage site. OGG1 recruitment facilitates the dissociation of DDB2. In the absence of downstream repair, DDB2 is retained longer at 8-oxoG sites requiring DDB1-Cul4A-RBX1 (CRL) mediated DDB2 dissociation. At actively transcribed regions, OGG1 can access the lesion independent of DDB2. 8-oxoG processing can lead to toxic BER intermediates that can act as a transcription block. Transcription-coupled repair (TCR) proteins, including XPA, participate in the repair of these BER intermediates. (See also Supplementary Movie ).

Article Snippet: Primary antibodies used: DDB2 (1:1000; abcam #ab181136), OGG1(1:1000; abcam #124741), Cul4A (1:1000; CST #2699 S), DDB1 (1:1000; Invitrogen #37-6200), XPC (1:1000; CST #12701 S), CSB (1:1000; abcam #ab96089), mCherry (1:1000; Abcam #ab167453), β-actin (1:30,000; Sigma #A2228).

Techniques: Expressing, Blocking Assay

Journal: Cancer Research

Article Title: Nuclear Receptor Coactivator NCOA3 Regulates UV Radiation–Induced DNA Damage and Melanoma Susceptibility

doi: 10.1158/0008-5472.can-20-3450

Figure Lengend Snippet: Figure 5. Effects of NCOA3 levels on UVR sensitivity in human melanoma cell lines. A, Representative images of immunofluorescence detection of NCOA3 and XPC in C8161.9 cells treated with vehicle (DMSO) or bufalin (quantitation provided in Supplementary Fig. S3F). B, Cell survival analysis of UVC-exposed C8161.9 cells (in triplicate). C, Cell survival analysis of UVC-exposed D04 cells (in triplicate). D, Cell survival analysis of UVC-exposed Ma-Mel-12 cells (in triplicate). E, Colony formation assay of UVC-exposed C8161.9 cells previously treated with vehicle (DMSO) or bufalin. F, Colony formation assay of UVC-exposed D04 cells previously treated with vehicle (DMSO) or bufalin. G, Colony formation assay of UVC-exposed Ma-Mel-12 cells previously treated with vehicle (DMSO) or bufalin. H, Effects of XPC cDNA overexpression on colony formation of UVC-exposed C8161.9 cells previously treated with bufalin (all assays performed in triplicate). , P < 0.05 denotes statistically significant differences compared with control. Scale bars, 20 mm.

Article Snippet: To overexpress pcDNA3, human NCOA3 cDNA or T960T NCOA3 polymorphism cDNA in NHEM Mel-F-Neo cells and human XPC cDNA (or a control plasmid expressing enhanced GFP, or EGFP) in C8161.9 cells, lipofectamine 2000–mediated transfections (9 mL) of (3 mg) plasmids expressing control pcDNA3 or EGFP, human XPC cDNA (Addgene#39204), human NCOA3 (Genecopoeia), or T960T were carried out following manufacturer’s instructions (Thermo Fisher Scientific).

Techniques: Quantitation Assay, Colony Assay, Over Expression, Control

Journal: Cancer Research

Article Title: Nuclear Receptor Coactivator NCOA3 Regulates UV Radiation–Induced DNA Damage and Melanoma Susceptibility

doi: 10.1158/0008-5472.can-20-3450

Figure Lengend Snippet: Figure 6. Role of differential NCOA3 levels in UV and melanoma susceptibility. A, Representative images of immunofluorescence detection of various proteins in NHEM cells transfected with control (pcDNA) or human NCOA3 cDNA plasmids (quantitation provided in Supplementary Fig. S8A). B, Representative images of immuno- fluorescence detection of NCOA3 and gH2AX in NHEM cells transfected with control (pcDNA) or human NCOA3 cDNA plasmids with UVC treatment (quantitation provided in Supplementary Fig. S8B). C, Prevalence of the T960T NCOA3 polymorphism in a control population (364 cases) versus a familial melanoma cohort (97 cases; left), and in the European (E; HapMap-CEU) versus Sub-Saharan African populations (S-S A; HapMap-YRI; right). D, Western blot analysis of NCOA3 and GAPDH in NHEM cells transfected with control (pcDNA), human T960T NCOA3 polymorphism cDNA (hT960T), or human wild-type NCOA3 cDNA (hNCOA3) plasmids (densitometric values provided were normalized to GAPDH). E, UVC sensitivity (in triplicate) of NHEM cells transfected with control (pcDNA), human T960T NCOA3 polymorphism cDNA (hT960T), or human wild-type NCOA3 cDNA (hNCOA3) plasmids by cell survival analysis. F, ELISA assay (in triplicate) of 6-4PP from UVC-exposed NHEM cells transfected with control (pcDNA), human T960T NCOA3 polymorphism cDNA (hT960T) or human wild-type NCOA3 cDNA (hNCOA3) plasmids. G, ELISA assay (in triplicate) of CPD from UVB-exposed NHEM cells transfected with control (pcDNA), human T960T NCOA3 polymorphism cDNA (hT960T), or human wild-type NCOA3 cDNA (hNCOA3) plasmids. , P < 0.05 denotes statistically significant differences compared with control. Scale bars, 20 mm.

Article Snippet: To overexpress pcDNA3, human NCOA3 cDNA or T960T NCOA3 polymorphism cDNA in NHEM Mel-F-Neo cells and human XPC cDNA (or a control plasmid expressing enhanced GFP, or EGFP) in C8161.9 cells, lipofectamine 2000–mediated transfections (9 mL) of (3 mg) plasmids expressing control pcDNA3 or EGFP, human XPC cDNA (Addgene#39204), human NCOA3 (Genecopoeia), or T960T were carried out following manufacturer’s instructions (Thermo Fisher Scientific).

Techniques: Transfection, Control, Quantitation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Catalytically Active Proteasomes Function Predominantly in the Cytosol

doi: 10.1074/jbc.m115.712406

Figure Lengend Snippet: FIGURE 3. Proteasome localization in cultured human cells. A, mammalian HEK293T cells were fractionated, and proteasome peptidase activity was determined in the nuclear and cytosolic fractions. Error bars represent S.E. from four independent experiments. Peptidase activity was detected solely in the cytoplasmic lysate, using either equal amount of protein (conc.) or proportional volumes of lysates (vol.) following fractionation. B, total, cytoplasmic, and nuclear fractions were prepared from HEK293T cells and separated in a native polyacrylamide gel. LLVY-AMC hydrolysis was examined in situ. A 5-fold higher loading of the nuclear extract (5 Nuc) was also examined. The positions of the single-capped and double-capped proteasomes are shown. C, protein extracts fromHEK293TcellswereseparatedbySDS-PAGEandtransferredtonitrocellulose,andthefiltersstainedwithPonceau-S.Adjustingthelysatestoequalprotein concentrations resulted in significant over-representation of nuclear proteins (lanes 1–3), as noted above (Fig. 2). Antibody reactions against tubulin (cytosolic) andhistoneH3(nuclear)showedthatthefractionationyieldednucleithatwerefreeofcytoplasmicproteins.Anasteriskinlane3identifiesanover-represented nuclear protein that cross-reacted with anti-Rpn12 antibody (seeD, below). D, immunoblot in C was reacted with antibodies indicated on theright. Proteasome subunits (19S and 20S subunits) and tubulin were detected only in the cytosol, and nuclear proteins (XPC, p53, and H3) were detected only in the nuclear fraction.

Article Snippet: Tubulin antibody was purchased from Life Technologies, Inc.; XPC antibody was purchased from Novus; proteasome and immunoproteasome antibodies were purchased from Enzo Biochem.

Techniques: Cell Culture, Activity Assay, Fractionation, In Situ, Western Blot

Journal: Nucleic Acids Research

Article Title: Single-molecule analysis of DNA-binding proteins from nuclear extracts (SMADNE)

doi: 10.1093/nar/gkad095

Figure Lengend Snippet: DNA-binding interactions of other DNA repair proteins. ( A ) The structure of eGFP-XPC (PDB codes 6CFI of Rad4 the yeast homolog to XPC and 4EUL). ( B ) A cartoon depiction of the DNA substrate used for XPC binding characterization, with UV damage sites shown in yellow and XPC binding shown in blue. Also shown is an example kymograph of eGFP-XPC binding and diffusing along the DNA in yellow. ( C ) CRTD analysis of XPC binding DNA with UV damage. ( D ) Distribution of motile and nonmotile XPC events. ( E ) An example MSD plot for analyzing XPC diffusion on DNA (D, in μm 2 /s). ( F ) Diffusion and α values for the diffusion of XPC on DNA. Event marked with asterisk was too short to determine an α value so it was defined as 1.0. ( G ) A structural model of APE1-tGFP from PDB code (5WNO and 4EUL). ( H ) Schematic and example kymograph of APE1 binding to DNA with nicks. ( I ) CRTD analysis of APE1 binding nicked DNA, with fit shown in blue. ( J ) A structural model of polβ-tGFP, taken from PDB codes (4KLO and 4EUL) and the tGFP modeled in. ( K ) Example schematics of polβ binding DNA containing nicks as well as a corresponding kymograph of an observation of polβ binding. ( L ) CRTD analysis of polβ binding nicked DNA, with the fit shown in blue. See .

Article Snippet: Primary antibodies used: PARP1 (1:100; abcam #ab227244), DDB2 (1:1000; abcam #ab181136), DDB1 (1:1000; Invitrogen #37-6200), XPC (1:1000; Novus #NB100-477) Polβ (1:1000; proteintech #18003-1-AP), OGG1 (1:1000; abcam #ab124741), and APE1 (1:100; Abcam #ab194).

Techniques: Binding Assay, Diffusion-based Assay