The Replicase Sliding Clamp Dynamically Accumulates behind Progressing Replication Forks in Bacillus subtilis Cells  Masayuki Su'etsugu, Jeff Errington  Molecular Cell  Volume 41, Issue 6, Pages 720-732 (March 2011) DOI: 10.1016/j.molcel.2011.02.024 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Localization of GFP-DnaN (A) Localization of GFP-DnaN in asynchronously replicating cells. Strain MS104 (gfp-dnaN) was grown in MMglyC medium at 30°C. The DAPI staining image of nucleoid DNA and the phase-contrast image are overlaid. Scale bar: 2 μm. (B) Localization of GFP-DnaN in the absence of replication. Strain MS114 (dnaB134ts, gfp-dnaN) was grown in MMglyC medium at 30°C and then shifted to 45°C for 2 hr. Cells were then immediately subjected to microscopic analysis. The phase-contrast image shows the outline of each cell. Scale bar: 2 μm. (C–E) After the completion of ongoing rounds of replication in (B), replication initiation was released on an agar pad at room temperature (25°C), and cells were analyzed by time-lapse microscopy. Typical still frames taken at 10 s intervals are shown in (C). Scale bar: 2 μm. Fluorescence signals in an area of the focus (indicated by white arrows) were quantified. An outline of the image is given in (D). As shown in (E), the ratio of the focus signal to the whole-cell signal was plotted over time. The dotted line represents the background value in the quantified area before focus formation. Molecular Cell 2011 41, 720-732DOI: (10.1016/j.molcel.2011.02.024) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Formation of GFP-DnaN Foci in Wild-Type Cells (A and B) Strain MS104 (gfp-dnaN) was grown in MMgly medium at 30°C and analyzed by time-lapse microscopy. Typical still frames and the signal ratio are shown as in Figure 1. Cell generating a single focus (5 s intervals) is shown in (A); in (B), cell generating two foci (foci b and c) and the disappearance of a previous focus (focus a) (10 s intervals). The phase-contrast image was taken, and cell outlines are shown by dotted lines. Scale bar: 2 μm. (C and D) Cellular content of DnaN. Strains 168 (wild-type) and MS104 (gfp-dnaN) were grown to A600 = 0.2 in the same condition as in (A) and (B). Portions of culture (50 μl) were subjected to western blot analysis using anti-DnaN antiserum (C). Amounts of wild-type DnaN and GFP-DnaN were deduced from the quantitative standard of purified His6-DnaN protein (His-DnaN). Asterisk (∗) indicates a nonspecific band. A portion of the same culture was subjected to cell counting by plating (D). The cellular contents of DnaN and GFP-DnaN were calculated based on the calculated molecular mass of the DnaN dimer (84 kDa) and Avogadro's number (6.02 × 1023 molecules/mol). See also Figure S1 for focus formation in cells bearing DnaX-YFP or PolC-GFP. Molecular Cell 2011 41, 720-732DOI: (10.1016/j.molcel.2011.02.024) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 DNA Loading Is Required for DnaN Accumulation (A and B) DnaG dependence of DnaN focus formation in outgrowing spores. Spores of strain MS242 (gfp-dnaN, amyE::PspacHY-dnaG, ΔdnaG) were geminated and outgrown in germination medium in the presence (A) or absence (B) of 1 mM IPTG. The phase-contrast image reveals the outline of each cell. Scale bar: 2 μm. (C–F) Localization of the dimerization-deficient DnaN mutant protein. Alignments of amino acid residues flanking the mutation sites in E. coli (Ec) and B. subtilis (Bs) DnaNs are shown in (C). Schematic view of this experiment is given in (D). Strains MS320 (amyE::Pxyl-yfp-dnaN) (E) and MS328 (amyE::Pxyl-yfp-dnaNmono) (F) were grown in MMgly medium in the presence of 0.5% xylose at 30°C. The phase-contrast image reveals the outline of each cell. Scale bar: 2 μm. (G and H) A decrease in the DnaG concentration slows the rate of DnaN accumulation. Strain MS242 (gfp-dnaN, amyE::PspacHY-dnaG, ΔdnaG) was grown in MMgly medium in the presence of 10 μM IPTG at 30°C. Focus generation was analyzed by time-lapse microscopy at 20 s intervals, as in Figure 1. An image of 5 s exposure was taken after the time lapse (long exposure), and cell outlines are shown by dotted lines. Scale bar: 2 μm. Molecular Cell 2011 41, 720-732DOI: (10.1016/j.molcel.2011.02.024) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Localization of DnaN Foci Relative to the Replication Fork (A) B. subtilis chromosomal loci chosen for analysis. The distance (kb) from oriC and the estimated time (min) of fork traverse are shown. (B and C) Strain MS137 (dnaB134ts, his12-dnaN) was grown at 30°C and then shifted to 45°C for 1.5 hr. After initiation release at 30°C for 10 min, 250 μg/ml rifampicin was added (time = 0). Cells were collected at the indicated times and subjected to marker frequency analysis to monitor the fork progression (B) and ChAP analysis (C). The experiment was repeated twice with different time points for different loci, and a representative data set is shown. (D–I) Strains MS157 (dnaN-mCherry, gfp-dnaC) (D–F) and MS110 (cfp-dnaN, dnaX-yfp) were grown in MMgly medium at 30°C. The images shown are: mCherry (D), GFP (E), overlay of mCherry (red) and GFP (green) (F), CFP (G), YFP (H), and overlay of CFP (blue) and YFP (yellow) (I). Scale bar: 2 μm. Molecular Cell 2011 41, 720-732DOI: (10.1016/j.molcel.2011.02.024) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Replication Arrest Disperses DnaN Foci (A) Model for translocation of the DnaN accumulation zone. (B–D) Strain MS104 (gfp-dnaN) was grown in MMgly medium at 30°C. Cells were taken before (−HPUra) and 2, 20, and 60 min after the addition of 47 μg/ml HPUra and subjected to microscopic analysis using 10 s exposure times (B). Two minutes after the addition of HPUra, time-lapse analysis using 200 ms exposure times was started (+HPUra) (C and D). A control analysis was done with no HPUra (−HPUra). Typical still frames, with phase-contrast image showing the outlines of each cell, are shown (C). Scale bar: 2 μm. The signal ratio of focus to whole cell was quantified for cells with a single focus (n = 10) (D), and the average was plotted with error bars representing standard deviations. To deduce a dispersion rate, the background ratio was subtracted (dashed line), and the data were fitted to an exponential curve (gray line). (E–G) Strains MS328 (amyE::Pxyl-yfp-dnaNmono) (E), MS151 (yfp-ssb) (F), and MS150 (gfp-dnaC) (G) were grown in MMgly medium (supplemented with 1% xylose for MS328) at 30°C and analyzed before (−HPUra) and 20 min after (+HPUra) the addition of 47 μg/ml HPUra. The phase-contrast image shows the outline of each cell. Scale bar: 2 μm. Molecular Cell 2011 41, 720-732DOI: (10.1016/j.molcel.2011.02.024) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 DnaN within Foci Undergoes a Continuous Unloading-Reloading Cycle (A–E) FRAP/FLIP behaviors of DnaN. Strain MS356 (amyE::Pxyl-yfp-dnaN, dnaN::Pspac-dnaN) was grown in MMgly medium in the presence of 0.01% xylose at 30°C and mounted on agar pads with no xylose. Cells with a single DnaN focus (A and B) or with two foci (C–E) were analyzed. Dotted circles indicate the bleached areas, and recovery was monitored at 20 s intervals. Typical still frames are shown (A and C). Total cellular fluorescence before (−20 s) or just after (0 s) bleaching was quantified, and the relative values are shown. Long exposure images (5 s) were taken at the end of each time-lapse series, and the outline of each cell is indicated. Scale bar: 2 μm. Relative fluorescence intensity of the bleached focus (B and D, blue circles) or the adjacent nonbleached focus in the same cell (red circles) were plotted over time. For cells with two foci, the average of 15 experiments is also shown (E), with error bars representing standard deviations. Five foci in nonbleached cells were also analyzed to obtain their fading rate during image acquisition (0.25% per acquisition as an average). The data were corrected using this rate. (F) Computer models for FRAP and FLIP behavior in cells with a single focus or two foci. DnaN molecules on (fON and nfON) or off (fOFF and nfOFF) DNA are modeled in the focus (circle) or cell (ellipse) areas, respectively. OFF molecules are allowed to diffuse. The equations used for the fluorescent molecules in the single focus model are shown. Laser treatment was modeled by changing fluorescent molecules (fON and fOFF) in the dotted circle area to nonfluorescent molecules (nfON and nfOFF). The simulation was performed using parameter values of Koff = 0.0028 s−1 for the single-focus model or 0.0057 s−1 for the two-foci model and Kpri = 0.008 μM/s for both models. Cyan and pink lines in (B) and (E) show the behaviors of fON in the simulations compared with the corresponding experimental data. (G) Computer simulation of the DnaN accumulation behavior. The Koff value was determined from the plateau level of the experimental data (0.0042 s−1). After the simulation, the signal ratio of focus to whole cell was plotted (cyan line) and was compared with the experimental data of Figure 2A (gray circles). Molecular Cell 2011 41, 720-732DOI: (10.1016/j.molcel.2011.02.024) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Formation of DnaN-Dependent Foci by GFP Fusions Bearing a CBS Peptide (A–C) Strains MS303 (amyE::Pxyl-cbs-gfp) (A), MS307 (amyE::Pxyl-gfp-cbs) (B), and MS304 (amyE::Pxyl-cbsmut-gfp) (C) were grown in MMglyC medium at 30°C in the presence of 1% xylose. The ratio of cells with foci (n = 250–300) is shown in each panel. Scale bar: 2 μm. (D–F) Colocalization of GFP-CBS with DnaN-mCherry. MS354 (amyE::PspacHY-gfp-cbs, dnaN-mCherry) was grown in MMglyC medium at 30°C in the presence of 1 mM IPTG. Shown are: the GFP image (D), the mCherry image (E), and overlay of the GFP (green) and the mCherry (red) images (F). Scale bar: 2 μm. (G–J) The DnaN dependence of focus formation by CBS-GFP. DnaA and DnaN were depleted as described previously (Meile et al., 2006). Stains MS111 (gfp-dnaN, dnaA::Pspac-dnaA) (G and H) and MS305 (amyE::Pxyl-cbs-gfp, dnaA::Pspac-dnaA) (I and J) were grown in LB medium at 30°C in the presence of 0.5 mM IPTG (G and I). After the removal of IPTG, incubation was continued for another 3 hr (H and J). For CBS-GFP expression, 1% xylose was added. The GFP image is overlaid with the phase contrast image (G and H). Scale bar: 2 μm. Molecular Cell 2011 41, 720-732DOI: (10.1016/j.molcel.2011.02.024) Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 41, 720-732DOI: (10.1016/j.molcel.2011.02.024) Copyright © 2011 Elsevier Inc. Terms and Conditions