1. Temporal Regulation of Topoisomerase IV Activity in E. coli
Olivier Espeli, Cindy Levine, Heide Hassing, Kenneth J. Marians 
Molecular Cell 
Volume 11, Issue 1, Pages (January 2003)
DOI: /S (03)

2. Figure 1 Activity of γE145A and τE145A during DNA Replication In Vitro
The activities of γE145A and τE145A were compared to that of the wild-type proteins in the M13Gori (A), rolling circle (B), and oriC (C) replication assays. Replication products were analyzed by denaturing alkaline agarose gel electrophoresis in (B) and by neutral agarose gel electrophoresis in (C). In (B) and (C), the total DNA synthesis is shown below each lane as [α-32P]dAMP incorporated into acid-insoluble product (pmol). In (C) “wt” and “E” refer to the wild-type and mutant versions, respectively, of τ and γ. LRI, late replication intermediate; FII, form II DNA (the decatenated product of the replication reaction). II:II dimers are the catenated product of the replication reaction.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 W3110dnaXE145A Exhibits a Severe Filamentation Phenotype
(A) Comparison of the growth of W3110 and W3110dnaXE145A in rich media at 37°C.
(B) Comparison of replication in W3110 and W3110dnaXE145A. Cells were grown at 37°C in rich media supplemented with 20 μg/ml thymine and [methyl-3H]thymidine (36 μCi/ml). Aliquots (200 μl) of cell culture were treated with trichloroacetic acid (to 5%), and acid-insoluble radioactivity was determined. The data shown represent an average of three independent experiments.
(C) Aliquots were removed from the cultures at the indicated times, chloramphenicol was added to 200 μg/ml, and the incubation continued for 5 min. The cells were then fixed, stained with DAPI, and visualized by fluorescence microscopy. The bar in (C) for the 2 hr time point of W3110dnaXE145A represents 8 μm.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Nucleoids from W3110dnaXE145A Contain Daughter Chromosomes that Have Not Been Decatenated
(A) Nucleoids purified from W3110dnaXE145A cells growing rapidly in early log phase were visualized by light microscopy before and after treatment with Topo IV.
(B) A histogram comparing the distribution of size of W3110dnaXE145A nucleoids before and after 1 and 2 hr of incubation with Topo IV, and after 2 hr of incubation in the absence of Topo IV, with those purified from W3110.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 ParC Localization Varies with the Growth Stage of the Cell
ParC and FtsZ were localized in W3110(λftsZ-gfp). ParC staining is shown in red in (A), (B), (E), and (F). FtsZ-GFP staining is shown in green in (A), (C), (E), and (G). DAPI staining is shown in blue in (A), (D), (E), and (H). (A) is the merge of (B)–(D), and (E) is the merge of (F)–(H). The locations of ParC and FtsZ-GFP were quantitated for cells of various average sizes in (I) (average size 2.2 μm), (J) (average size 2.9 μm with two ParC foci), (K) (average size 2.9 μm with three ParC foci), and (L) (average size 4 μm). Distances were measured from one pole of the cell and plotted against overall cell length. ParC mid, left, and right refer to the location of ParC foci in the cell as disposed about the FtsZ-GFP ring defining the middle (mid) of the cell. ParC left 1, left 2, right 1, and right 2 refer to cells where there are multiple ParC foci flanking the cell center. The white bar in (A) represents 3 μm. In (M) these data are summarized in a schematic format. Stippled blue ovoids are nucleoids, small red spheres are ParC foci, green bars are FtsZ rings, and dashed green bars represent the site of future FtsZ rings. The distribution of each cell type in the culture is also indicated.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 ParC Localizes to the Replication Factory
α, β, and τ/γ were localized in W3110 cells (A–R). (A)–(E) are merged images of staining for β (red) and DIC images of the cells. (F)–(I) are merged images of staining for τ/γ (red) and DIC images of the cells. (J)–(Q) are pairs of images showing staining for α (red) on the left and a merge of the α stain with a DAPI stain of the DNA (independent image not shown) on the right. Quantitation of β position is shown in (R). Distances were measured from one pole of the cell and plotted against overall cell length. Beta left 1, left 2, right 1, and right 2 refer to the relative positions of the β foci about the cell center (β mid). The bar in (O) represents 2 μm. ParC and β colocalize ([S], i–iv). Four sets of images are shown (i–iv). The panel on the left shows the ParC signal (green), the center panel shows the β signal (red), and the panel on the right shows the merge. All images are also merged with a DIC image of the cells. The bar in (I) represents 2 μm.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 ParE Is Found in DNA-free Spaces in the Cell
ParE was localized in W3110. Representative fields are shown in (A)–(F). ParE staining is in red in (A), (B), (D), and (E). DAPI staining is in green in (A), (C), (D), and (F). (A) is the merge of (B) and (C), and (D) is the merge of (E) and (F). The white bar in (A) represents 3 μm.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7 ParC Is Mislocalized in W3110dnaXE145A
HE and Topo IV subunits were localized in either W3110dnaXE145A (A–C) or W3110dnaXE145A(λftsZ-gfp) (E and G).
(A and B) Replication factories are present in filaments of W3110dnaXE145A. The locations of α (A) and τ/γ (B) were determined. Antibody staining is colored red, and all images are merges with DIC images of the cells. The bar in (A) represents 3 μm.
(C) α and τ/γ colocalize. A merged image of DAPI-stained DNA (blue), staining for α (red), and staining for τ/γ (green) is shown.
(D) Intensity profile as a function of distance from one pole of the cell of the three color signals shown in the image in (C).
(E) ParC is mislocalized. A merged image of DAPI-stained DNA (blue), staining for ParC (red), and the signal for FtsZ-GFP (green).
(F) Intensity profile of the three color signals shown in the image in (E).
(G) ParE localization is maintained. A merged image of DAPI-stained DNA (blue), staining for ParE (red), and the signal for FtsZ-GFP (green).
(H) Intensity profile of the three color signals shown in the image in (G).
Molecular Cell  , DOI: ( /S (03) )

9. Figure 8 Topo IV Activity Is Manifest Late in the Cell Cycle
PC2 and PC2nalr cultures were synchronized by a triple temperature shift.
(A) Norfloxacin-induced cell killing, [3H]thymidine uptake and cell number were determined.
(B) TUNEL was performed using cells removed from the synchronized cultures at the indicated times. TUNEL staining is shown in red and DAPI staining in green.
Molecular Cell  , DOI: ( /S (03) )