plx304 targ1 wt  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: TARG1 protects against toxic DNA ADP-ribosylation

doi: 10.1093/nar/gkab771

Figure Lengend Snippet: TARG1 removes thymidine-linked ADP-ribose from DNA and confers resistance to DarT toxicity in bacteria. ( A ) Structural comparison between TaqDarG-macrodomain (orange) bound to ADP-ribose (red) and TARG1 (light blue) with a covalent lysyl-ADP-ribose linkage (dark blue). To the right is a detailed view of the DarG catalytic lysine 80 (orange sticks) and TARG1 catalytic lysine 84 (blue sticks). ( B ) UV detection of DarT ADP-ribosylated DNA oligonucleotide de-ADP-ribosylation reactions with TaqDarG-macrodomain and TARG1. ( C ) Bacterial DarT toxicity rescue assay in BL21 DE3 using pBAD DarT and pET encoding DarG-macrodomain WT, DarG-macrodomain K80A, TARG1 WT or TARG1 K84A. pBAD expression is controlled with glucose or arabinose and pET expression is controlled with IPTG.

Article Snippet: Plasmids used here include: pBAD33-V5-DarT ( Thermus aquaticus ), pET28a-6xHis-TARG1-WT, pET28a-6xHis-TARG1-K84A, pET28a-6xHis-DarG-WT-1–155aa ( Thermus aquaticus ), pET28a-6xHis-DarG-K80A-1–155aa ( Thermus aquaticus ), pLIX_403-GFP-DarT-WT ( Thermus aquaticus ), pLIX_403-GFP-DarT-E160A ( Thermus aquaticus ), pLX304-TARG1-WT, pLX304-TARG1-K84A. pLIX_403 and pLX304 were gifts from David Root (pLIX_403 Addgene: #41395, pLX304 Addgene: #25890)

Techniques: Rescue Assay, Expressing

Journal: Nucleic Acids Research

Article Title: TARG1 protects against toxic DNA ADP-ribosylation

doi: 10.1093/nar/gkab771

Figure Lengend Snippet: DarT induces the DDR in TARG1-deficient cells and limits DNA replication ( A ) Representative images of clonogenic survival in U-2 OS WT or TARG1 KO cells expressing pLIX_403 GFP-DarT WT or GFP-DarT E160A. pLIX_403 expression was controlled by doxycycline. ( B ) U-2 OS WT or TARG1 KO cells treated with HU (2 mM, 24 h), CPT (1 μM, 1 h), MMS (2 mM, 1 h), DarT WT (24 h), DarT E160A (24 h). Levels of DDR proteins KAP1 pS824, RPA2 pS4/8, γH2AX were analysed in response to the aforementioned genotoxins. ( C ) QIBC analysis of EdU incorporation in U-2 OS WT and TARG1 KO cells in response to the genotoxins found in Figure 2B. Prior to fixation, cells were treated with EdU (10 μM, 30 min). Red bar represents the mean EdU intensity for each population and each point represents the mean EdU intensity for an individual nuclei. ( D ) U-2 OS WT or TARG1 KO cells expressing either DarT WT or TARG1 KO for 24 h were pulse labeled with CldU followed by IdU for 25 min each, as indicated by the labeling protocol, top. IdU track lengths were measured for 100 fibers per condition. Statistical tests were performed using ANOVA where ns is not significant and **** is P < 0.0001. In the box plots, the box extends from the 25th (lower edge) to 75th (higher edge) percentiles and the line within the box represents the median. Whiskers extend to the minimum and maximum values recorded. Representative fiber images can be found in . ( E ) U-2 OS TARG1 KO cells expressed GFP-DarT for 24 h and were treated with EdU (10 μM, 30 mins) prior to pre-extraction and fixation. Following a Click-iT reaction to visualise EdU, cells were then immunostained for GFP and chromatin-bound RPA2. Scale bar 20 μm. ( F ) QIBC analysis of chromatin-bound RPA2 of U-2 OS TARG1 KO cells in response to the genotoxins found in Figure 2B. Blue bar represents the mean RPA2 intensity for each population and each point represents the mean chromatin-bound RPA2 intensity for an individual nuclei.

Article Snippet: Plasmids used here include: pBAD33-V5-DarT ( Thermus aquaticus ), pET28a-6xHis-TARG1-WT, pET28a-6xHis-TARG1-K84A, pET28a-6xHis-DarG-WT-1–155aa ( Thermus aquaticus ), pET28a-6xHis-DarG-K80A-1–155aa ( Thermus aquaticus ), pLIX_403-GFP-DarT-WT ( Thermus aquaticus ), pLIX_403-GFP-DarT-E160A ( Thermus aquaticus ), pLX304-TARG1-WT, pLX304-TARG1-K84A. pLIX_403 and pLX304 were gifts from David Root (pLIX_403 Addgene: #41395, pLX304 Addgene: #25890)

Techniques: Expressing, Labeling

Journal: Nucleic Acids Research

Article Title: TARG1 protects against toxic DNA ADP-ribosylation

doi: 10.1093/nar/gkab771

Figure Lengend Snippet: DarT induces the DDR at sites of DNA replication ( A ) Representative images of U-2 OS TARG1 KO cells expressing either GFP-DarT WT or E160A for 24 h were pre-extracted and immunostained for GFP, γH2AX and PCNA. Nuclear DNA was counterstained with DAPI. Scale bar 20 μm. ( B ) Representative images of U-2 OS TARG1 KO cells expressing either GFP-DarT WT or E160A for 24 h were pre-extracted and immunostained for GFP, RPA2 pS4/8 and PCNA. Nuclear DNA was counterstained with DAPI. Scale bar 20 μm. ( C ) Workflow for QIBC analysis. ( D ) QIBC analysis of asynchronous U-2 OS TARG1 KO cells expressing GFP-DarT WT for 24 h. Cells were pre-extracted, fixed and immunostained as in A. QIBC was used to record total DAPI intensity, mean PCNA intensity and mean γH2AX intensity per nucleus for >1000 cells. DAPI and PCNA intensities for individual cells were used to generate the scatter plot and γH2AX intensity was used to colour points. ( E ) QIBC analysis of γH2AX in TARG1 KO cells in response to the genotoxin treatments found in Figure . Blue bar represents the mean γH2AX intensity for each population and each point represents the mean γH2AX intensity for an individual nuclei. ( F ) U-2 OS TARG1 KO cells were treated and analysed as in (D) and immunostained as in (B). ( G ) QIBC analysis as in E and immunostained as in (B).

Article Snippet: Plasmids used here include: pBAD33-V5-DarT ( Thermus aquaticus ), pET28a-6xHis-TARG1-WT, pET28a-6xHis-TARG1-K84A, pET28a-6xHis-DarG-WT-1–155aa ( Thermus aquaticus ), pET28a-6xHis-DarG-K80A-1–155aa ( Thermus aquaticus ), pLIX_403-GFP-DarT-WT ( Thermus aquaticus ), pLIX_403-GFP-DarT-E160A ( Thermus aquaticus ), pLX304-TARG1-WT, pLX304-TARG1-K84A. pLIX_403 and pLX304 were gifts from David Root (pLIX_403 Addgene: #41395, pLX304 Addgene: #25890)

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: TARG1 protects against toxic DNA ADP-ribosylation

doi: 10.1093/nar/gkab771

Figure Lengend Snippet: DarT induces ADP-ribose foci in S-phase cells with no ADP-ribosylation detectable by western blot. ( A ) U-2 OS TARG1 KO cells treated with HU (2 mM, 24 h), CPT (1 μM, 1 h), MMS (2 mM, 1 h), DarT (24 h), DarT E160A (24 h). Levels of ADP-ribosylation were assessed using the CST poly/mono ADP-ribose antibody in response to the aforementioned genotoxins. ( B ) QIBC analysis of ADP-ribosylation in TARG1 KO cells in response to the genotoxin treatments in A. Blue bar represents the mean ADP-ribosylation intensity for each population and each point represents the mean ADP-ribosylation intensity for an individual nuclei. ( C ) Representative images of U-2 OS TARG1 KO cells expressing either GFP-DarT WT or E160A for 24 h or cells treated with MMS (2 mM, 1 h) were pre-extracted and immunostained for GFP, ADP-ribosylation, PCNA and nuclear DNA was counterstained with DAPI. Scale bar 20 μm. ( D ) Magnified view of the region enclosed by the white square in (B) for DarT and MMS-treated cells. Scale bar 20 μM. ( E ) QIBC analysis of asynchronous U-2 OS TARG1 KO cells expressing GFP-DarT WT for 24 h or treated with MMS (2 mM, 1 h). Cells were pre-extracted, fixed and immunostained as in (C). QIBC was used to record total DAPI intensity, mean PCNA intensity and mean ADP-ribosylation intensity per nucleus for >1000 cells. DAPI and PCNA intensities for individual cells were used to generate the scatter plot and ADP-ribosylation intensity was used to color points.

Article Snippet: Plasmids used here include: pBAD33-V5-DarT ( Thermus aquaticus ), pET28a-6xHis-TARG1-WT, pET28a-6xHis-TARG1-K84A, pET28a-6xHis-DarG-WT-1–155aa ( Thermus aquaticus ), pET28a-6xHis-DarG-K80A-1–155aa ( Thermus aquaticus ), pLIX_403-GFP-DarT-WT ( Thermus aquaticus ), pLIX_403-GFP-DarT-E160A ( Thermus aquaticus ), pLX304-TARG1-WT, pLX304-TARG1-K84A. pLIX_403 and pLX304 were gifts from David Root (pLIX_403 Addgene: #41395, pLX304 Addgene: #25890)

Techniques: Western Blot, Expressing

Journal: Nucleic Acids Research

Article Title: TARG1 protects against toxic DNA ADP-ribosylation

doi: 10.1093/nar/gkab771

Figure Lengend Snippet: Detection of thymidine-linked ADP-ribosylation in human genomic DNA. ( A ) U-2 OS TARG1 KO cells expressing GFP-DarT (24 h) or treated with MMS (2 mM, 1 h) were also treated with olaparib (10 μM, 24 h) or veliparib (10 μM 24 h). Levels of KAP1 pS824, RPA2 pS4/8, γH2AX and ADP-ribosylation were assessed. ( B ) Representative images of U-2 OS TARG1 KO cells treated as in (A). Cells were pre-extracted and immunostained for GFP, ADP-ribosylation and chromatin-bound RPA2. Scale bar 10 μm. ( C ) U-2 OS WT or TARG1 KO cells treated with HU (2 mM, 24 h), CPT (1 μM, 1 h), MMS (2 mM, 1 h), DarT WT (24 h), DarT E160A (24 h). Genomic DNA was extracted and dotted onto nitrocellulose membranes and immunoblotted for dsDNA or ADP-ribosylation using the CST poly/mono ADP-ribose antibody. ( D ) Genomic DNA from U-2 OS TARG1 KO cells expressing DarT was extracted as in (C). DNA was incubated with either 1 μM TaqDarG-macrodomain WT or TARG1 WT recombinant proteins in vitro. DNA was then immunoblotted as in (C).

Article Snippet: Plasmids used here include: pBAD33-V5-DarT ( Thermus aquaticus ), pET28a-6xHis-TARG1-WT, pET28a-6xHis-TARG1-K84A, pET28a-6xHis-DarG-WT-1–155aa ( Thermus aquaticus ), pET28a-6xHis-DarG-K80A-1–155aa ( Thermus aquaticus ), pLIX_403-GFP-DarT-WT ( Thermus aquaticus ), pLIX_403-GFP-DarT-E160A ( Thermus aquaticus ), pLX304-TARG1-WT, pLX304-TARG1-K84A. pLIX_403 and pLX304 were gifts from David Root (pLIX_403 Addgene: #41395, pLX304 Addgene: #25890)

Techniques: Expressing, Incubation, Recombinant, In Vitro