Journal: Nucleic acids research

Article Title: CSB interacts with SNM1A and promotes DNA interstrand crosslink processing.

doi: 10.1093/nar/gku1279

Figure Lengend Snippet: Figure 1. CSB and SNM1A interact. (A) Yeast two-hybrid interaction. The SNM1A-GAL4(AD) construct was cotransformed into NMY32 yeast with the indicated LexA(DBD) fusion construct (the pLEXA plasmids). The transformation mix was then spotted and allowed to grow on normal medium (SD-LW) or selection medium lacking histidine (SD-HLW) or histidine and adenine (SD-AHLW). Full-length CSB (CSBfl) and the 306 amino acid C- terminal CSB fragment (CSBc), as well as Lamin C and p53, were tested to determine interaction specificity. (B) Purified recombinant HA-tagged CSB and N-SNM1A proteins. Purified recombinant HA-CSB (300 ng, left) and N-SNM1A (300 ng, right) were loaded and separated on an 8% or 10% Tris–glycine–SDS-polyacrylamide gel, respectively. Proteins (designated by arrow) were then visualized using a Pierce Silver Stain Kit. Protein standards are indicated in kDa. (C) CSB physically interacts with the SNM1A nuclease fragment. N-SNM1A (500 ng) was incubated with or without HA-CSB (500 ng) in the presence of -HA magnetic beads. Bead-bound material was captured, washed and analyzed by silver staining after separation on an SDS- polyacrylamide gel. The positions of the CSB and SNM1A proteins are designated, as are the BSA, IgG-Heavy (H) and IgG-Light (L) bands intrinsic to the reaction mixture. I, 1/20th of the initial reaction mixture or input; W, material recovered from the third wash; IP, bead-bound, IP material. Protein standards are indicated in kDa. (D) Co-purification of endogenous CSB and SNM1A from human cell extracts. Whole cell extracts were prepared from HeLa cells, either without (-) or 0.5 or 2 h post-treatment with trioxsalen/UVA. Extracts were then incubated with -CSB antibody or mouse IgG, and subsequently with protein A/G magnetic beads. Bead-bound IP material was captured, washed, extracted with SDS sampling buffer and subjected to western blot analysis. The positions of CSB and SNM1A are shown, as are the protein standards in kDa. Bar graph indicates the relative SNM1A:CSB ratio (average and standard deviation of three independent experiments), with the untreated (-) sample set as 1. (E) Expression of CSB and SNM1A is unchanged following trioxsalen/UVA treatment. Whole cell extracts were prepared from HeLa cells, either without (-) or 0.5 or 2 h post-treatment with trioxsalen/UVA. Extracts were immediately subjected to western blot analysis for CSB, SNM1A or GAPDH, as designated. Bar graph reports the relative expression for CSB/GAPDH or SNM1A/GAPDH, as determined from three independent experiments.

Article Snippet: The SNM1A coding region was amplified using the primers SNM1A XhoI-Fw and SNM1A BamHI-Rv, and a cDNA template obtained from Origene (SC322694; Rockville, MD, USA).

Techniques: Construct, Transformation Assay, Selection, Purification, Recombinant, Silver Staining, Incubation, Magnetic Beads, Copurification, Sampling, Western Blot, Standard Deviation, Expressing

Journal: Nucleic acids research

Article Title: CSB interacts with SNM1A and promotes DNA interstrand crosslink processing.

doi: 10.1093/nar/gku1279

Figure Lengend Snippet: Figure 2. CSB modulates SNM1A exonuclease activity. Single-stranded or double-stranded 3′-end labeled 61-mer (A) or 21-mer (B) oligonu- cleotides were incubated with SNM1A (+; fixed concentration of 0.8 nM), and increasing concentrations of CSB where indicated (0.8–50 nM), for 30 min at 37◦C. Reactions were resolved on a denaturing gel, and images were captured during phosphorimager analysis (shown is a representative gel from three independent experiments). Note that the non-labeled strand of the double-stranded substrate is blocked at its 5′-end with a biotin moi- ety (B) to prevent competing digestion of this strand.

Article Snippet: The SNM1A coding region was amplified using the primers SNM1A XhoI-Fw and SNM1A BamHI-Rv, and a cDNA template obtained from Origene (SC322694; Rockville, MD, USA).

Techniques: Activity Assay, Labeling, Incubation, Concentration Assay

Journal: Nucleic acids research

Article Title: CSB interacts with SNM1A and promotes DNA interstrand crosslink processing.

doi: 10.1093/nar/gku1279

Figure Lengend Snippet: Figure 4. Response of SNM1A to site-specific DNA ICLs. (A) Quan- tification of the SNM1A response to laser/trioxsalen, and the effects of -amanitin on protein recruitment/retention. pSNM1A-GFP was trans- fected into HeLa cells, which were pre-treated with 6 M trioxsalen ± 20 M -amanitin as indicated, before targeted laser irradiation. The graph shows the RFI of SNM1A-GFP at the microirradiated area relative to unirradiated (background) parts of the nucleus. Each data point is de- rived from at least 16 independent cells from two independent experiments. Error bars indicate SEM. See Supplementary Figure S5A for represen- tative images. (B) SNM1A and CSB co-localize to sites of DNA ICLs in live human cells. HeLa cells were co-transfected with pSNM1A-GFP and pCSB-mCherry, and co-expressing cells were identified 20–24 h post- transfection. Cells were then microirradiated at the indicated region (yel- low box) under the conditions specified. Images of a representative sin- gle live cell are shown, along with the merge. (C) CSB coordinates the SNM1A response to DNA ICLs. CSB-deficient (CS1AN-vector) or CSB complemented (CS1AN-CSB) cells were transfected with pSNM1A-GFP and treated with 6 M trioxsalen for 30 min prior to targeted microirra- diation. See panel A for information about the graph, and Supplementary Figure S5B for representative images.

Article Snippet: The SNM1A coding region was amplified using the primers SNM1A XhoI-Fw and SNM1A BamHI-Rv, and a cDNA template obtained from Origene (SC322694; Rockville, MD, USA).

Techniques: Irradiation, Transfection, Expressing, Plasmid Preparation