R-Loop Formation, ssDNA Patches and Switching to IgA in WT and RNaseH1-Expressing CH12F3-2 Cells.

<p>A) Two CH12F3-2 control clones and two CH12F3-2 clones expressing RNaseH1 (hRH1) transfected with a hRH1 expression vector. Western blots were performed for RNaseH1 and β-actin. B) Switch region R-loop formation in control and RNaseH1-expressing CH12F3-2 cells. Extracted genomic DNA from <i>in situ</i> bisulfite treated-CH12F3-2 clones were subjected to PCR amplification using a standard forward primer and a reverse primer that preferentially binds to bisulfite-converted dCs on the top strand 5′ of the μ switch region to specifically amplify bisulfite converted products/R-loops (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#s4" target="_blank">Materials and Methods</a>). A hypothetical sequence in which all the dCs are converted to dUs is shown on the bottom for reference. The location of the reverse converted primer is depicted as a box. C) R-loop length as expressed as the mean length of contiguous converted dCs in control and RNaseH1-expressing CH12F3-2 cells. Data derived from B. Statistical analysis were conducted using the Student's two tailed t-test * = P = 0.016. D) ssDNA patch frequency, obtained from amplifying the switch region with unconverted primers, shown as ssDNA frequencies in control and RNaseH1-expressing CH12F3-2 cells before (Unstim) and after CSR-stimulation (Stim and hRH1) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Ramachandran1" target="_blank">[46]</a>. ssDNA frequency also analyzed in association with sequences that harbour R-loops (Cont with R-loop and hRH1 with R-loop). E) CSR to IgA in stimulated control and RNaseH1-expressing CH12F3-2 cells. ns = not significant. F) Mutation analysis in WT and hRH1 CH12F3-2 clones of the 5′Sμ region. Left Panel: Mutations are depicted at dC (opened triangle) or dA (closed triangle) along the 5′Sμ region. Middle Panel: mutation spectrum represented for WT and hRH1 samples. Right Panel: Mutation frequencies for WT and hRH1 samples (no significant difference observed).</p

ssDNA Frequencies at Non-Ig Sequences in LPS-Stimulated <i>Ex Vivo</i> Murine B Cells.

<p>A) ssDNA frequencies at the non-transcribed <i>CD4</i> gene, and other indicated genes that are transcribed in B cells. The genes are ordered from Spt5^hi (left) to Spt5^lo (right) and the number of αSpt5 TPM (tags per million sequences) obtained from Pavri <i>et al.</i> (see Table S3 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Pavri1" target="_blank">[3]</a>) is indicated above each gene, which correlates with Spt5 occupancy. B) ssDNA frequency plotted against the mutation frequency of each gene examined in (A). Mutation frequencies for each gene were obtained from Liu <i>et al... </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Liu1" target="_blank">[38]</a> (black symbols) and Pavri <i>et al... </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Pavri1" target="_blank">[3]</a> (grey symbols). C) Left Panel: ssDNA frequencies at the <i>Btg1</i> gene in pre-B cells, unstimulated B cell and LPS-stimulated B cells. Statistical analysis were performed using the Student's t-test (** = P = 0.0039 comparing ssDNA frequencies at the <i>Btg1</i> gene in pre-B cells and LPS-stimulated B cells). Right Panel: ssDNA patches depicted along the <i>Btg1</i> gene with unique AID-induced mutations. ssDNA patches observed in pre-B cells (open box), unstimulated mature B cells (grey box), and LPS-stimulated B cells (black box) are shown. Unique point mutations depicted at dC (open triangles) or dA (closed triangles) are shown and were obtained from Liu <i>et al... </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Liu1" target="_blank">[38]</a>. The location and strand distribution of WRC motifs are depicted as lines along the <i>Btg1</i> gene at the top of this panel.</p

ssDNA Frequencies during Induction of CSR in <i>Ex Vivo</i> Murine B Cells.

<p>A) ssDNA patches depicted 3′ of the JH4-region and 5′ of the μ switch region in <i>ex vivo</i> mouse B cells. ssDNA patches observed in unstimulated B cells (grey box), and LPS-stimulated B cells for 48 hours (black box) are shown. Patches are depicted as bars and are distributed to depict top- or bottom-strandedness. Wagon-wheels depict the number of ssDNA patches per sequence for both regions examined under unstimulated or LPS stimulated conditions. B) ssDNA patch lengths of the mouse V-region and 5′μ switch region. Median patch lengths depicted by a black line. C) ssDNA frequencies observed within murine <i>CD4</i> and the 3′JH4 region (left panel) and 5′Sμ (right panel) in unstimulated (open bar), LPS-stimulated (grey bar), and IgM and α-CD40 stimulated (dark grey bars) <i>ex vivo</i> mouse B cells. Statistical analyses were performed using the Student's t-test (Left Panel: ** = P = 0.002 comparing ssDNA frequencies at the CD4 gene and unstimulated 3′JH4 region; * = P = 0.021 comparing ssDNA frequencies at the V-region between unstimulated and LPS-stimulated B cells; Right Panel: * = P = 0.039 comparing ssDNA frequencies at the CD4 gene and the unstimulated 5′Sμ * = P = 0.036 comparing ssDNA frequencies at the 5′Sμ region between unstimulated and LPS-stimulated B cells). D) ssDNA frequencies at the 5′μ switch in CH12F3-2 cells and primary mouse B cells that were untreated or treated with Camptothecin (Campt) or Actinomycin D (Act-D) for 24 hrs. Since no ssDNA patches were observed in the Campt-treated CH12F3-2 cells, the data is presented as if one dC were converted to dT Statistical analysis were performed using the Student's t-test (* = P = 0.023 comparing CH12F3-2 no drug and Act-D LD50; * = P = 0.026 comparing Primary Mouse B cells LPS stim and LPS CPT LD50).</p

ssDNA Patches in the V-Region and GFP Transgene in Ramos Cells.

<p>A) Location and lengths of patches identified in the V-region and GFP transgene. Patches are depicted as black bars and distributed to depict top- or bottom-strandedness. Wagon-wheels depict the number of ssDNA patches per sequence for each gene. B) Cluster plot of patch lengths of ssDNA identified at the V region and GFP genes. Black line depicts the median patch size. C) Strand bias as an expression of ssDNA frequencies on the top strand divided by total ssDNA frequencies for each Ramos clone at the V-region and GFP transgene. A value of 0.5 suggests no strand bias. D) Mutation frequencies at the V-region and GFP genes for individual clones are reported. Statistical analysis was performed using the Mann-Whitney test (** = P = 0.0061). Values for V-region mutation frequencies were obtained from 2 Ramos clones for this study, as well as 5 Ramos clones from previous analyses <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Zhang1" target="_blank">[28]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Parsa1" target="_blank">[65]</a>. E) ssDNA frequencies at the V-region and GFP genes for individual clones. Statistical analyses were performed using the Mann-Whitney test (** = P = 0.0013). F) Linear regression analysis depicting ssDNA frequencies plotted against mutation frequencies for the GFP gene for 12 individual clones (GFP = open circles; VPS GFP = light grey circles; VPL GFP = dark grey circles; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518.s002" target="_blank">Figure S2C</a>). X and Y axes are plotted as linear values. G) Quantitative RT-PCR analyses for GFP expression was performed on the clones in which the GFP gene mutated at high rates (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518.s002" target="_blank">Figure S2C</a>; VPL-6) or low rates (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518.s002" target="_blank">Figure S2C</a>; GFP-14). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen-1002518-g001" target="_blank">Figure 1F</a>. The expression of GFP was plotted relative to GAPDH expression. The relative GFP expression levels of one replicate of clone GFP-14 was set to 1. Statistical analysis was performed using the Student's t-test (*** = P = 0.0009). H) ssDNA frequencies at the V-region in Ramos cells treated with the transcription elongation inhibitor, actinomycin D, or the topoisomerase I inhibitor, camptothecin, for 24 hrs with varying concentrations of inhibitors (LD25 and LD50). Act-D = Actinomycin D; Campt = Camptothecin. Statistical analysis was performed using the Student's t-test (* = P = 0.0115 comparing ssDNA frequencies in Act-D and untreated cells; * = P = 0.0108 comparing ssDNA frequencies in Campt and untreated cells).</p

ssDNA Patch Formation and Negative Supercoiling in <i>E. coli</i>: Wild-Type BL21(DE3) versus TopA Deficient VS111(DE3).

<p>A) p-GEX5.3-hAID(T27N) plasmid topology was determined for uninduced and 1 hr IPTG-induced bacterial strains by chloroquine agarose gel analysis for plasmids derived from wildtype BL21(DE3) and TopA-deficient VS111(DE3) bacterial strains. Highly-negative supercoiled DNA (HNSC) runs faster than its less supercoiled counterpart. B) ssDNA frequencies in uninduced and IPTG-induced bacterial strains. White bars indicate no IPTG treatment, black bars indicated IPTG treatment. Statistical analysis was performed using the Student's t-test (* = P = 0.0446 comparing ssDNA frequencies in uninduced and IPTG-induced BL21 cells; ** = P = 0.0059 comparing ssDNA frequencies in uninduced and IPTG-induced VS111 cells; ** = P = 0.0018 comparing ssDNA frequencies in IPTG-induced BL21 and VS111). C) ssDNA patch lengths in non-IPTG induced (open symbols) and IPTG induced (closed symbols) BL21 and VS111 bacterial strains. D) The location, lengths and strand distribution of ssDNA patches revealed in the p-GEX5.3-hAID(T27N) plasmid by <i>in situ</i> bisulfite treatment in uninduced (grey) and IPTG-induced (black) BL21(DE3) and VS111(DE3)[TopA mutant]. E) Schematic of the p-GEX5.3-hAID(WT)KanS vector used for the kanamycin reversion analysis. Kanamycin sensitivity is rendered by a proline (CCA) at position 94 of the kanamycin protein. C to T mutation of either the first or second C of the codon results in a serine or a leucine amino acid, respectively, which confers kanamycin resistance. F) AID-induced mutation frequencies of the kanamycin gene in BL21, MG1655 and TopA-deficient VS111 bacterial strains. Values were calculated after subtracting background mutation frequencies obtained from the mutation analysis of the p-GEX5.3KanS vector controls. EV = empty vector control; AID = AID expression vector (E). Statistical analysis were conducted using the student's two tailed t-test (*** = P = 0.0001 comparing BL21 EV and BL21 AID; *** = P = 0.0006 comparing MG1655 AID and VS111 AID; *** = P = 0.0008 comparing VS111 EV and VS111 AID).</p