Figure 9: Dumbbell-Shaped Nucleoids are Intermediates of Chromosome SegregationCompact nucleoids were purified from C600 and C600parC1215 and visualized by fluorescence microscopy after staining with DAPI. Two morphologically distinct classes are present in nucleoids purified from E.coli cells: (A) Singlets and (B) dumbbells.
Figure 10: Filaments of MreBElectron microscopic analysis of MreB filaments. MreB was diluted to a concentration of either 100 nM (D and E), 1 μM (C, F, and G), or 4 μM (H) and polymerized at 37 °C for either 5 min (C) or 30 min (D-H). Filaments were visualized by electron microscopy. (I) Distribution of the length of the filaments formed at the indicated concentrations of MreB.
Figure 11: The Oligomeric State of MreB Determines its Effect on Topo IV Decatenation Activity(A) Two sets of reactions were performed. In one set (lanes marked "M"), monomeric MreB was introduced into Topo IV kDNA decatenation reactions. kDNA is mitochondrial DNA obtained from trypanosomes such as Crithidia fasciculata and consists of large networks (~5000) of mostly covalently closed, catenated minicircle. Because of their high molecular weight, these networks are unable to enter agarose gels. However, type II topoisomerases such as Topo IV and can unlink the minicircles that can then be detected by agarose gel electrophoresis. In the second set of reactions (lanes marked "P"), the indicated amounts of MreB were pre-incubated for 30 min at 37 °C to form filaments prior to the addition of Topo IV. (B) Quantification of (A).
Figure 12: Interaction Between MreB and Topo IV May Help Coordinate Late Cell Cycle Events(1) Topo IV activity is spatially and temporally regulated during the bacterial cell cycle. (2) Active Topo IV is assembled only late in the cell cycle. Meanwhile, remodeling of MreB filaments during the cell cycle causes the accumulation of a ring of polymerized MreB at the cell center. (3) Polymerized MreB stimulates the decatenation activity of Topo IV. (4) MreB filaments are further remodeled to facilitate their segregation to the two daughter cells. This remodeling produces monomeric MreB. (5) Inhibition of Topo IV activity by monomeric MreB restores a state of low Topo IV activity at the onset of a new cell cycle.
Figure 13: The MinCDE System is the Target of the DNA Decondensation CheckpointMinD-GFP, which displays a pole-to-pole oscillation in WT cells, does not oscillate in the dnaXE145A strain (this mutation delocalizes Topo IV, leading to promiscuous Topo IV activity, decondensation of the nucleoid, and activation of the cell division checkpoint). (A) are wild-type cells; (B) are filaments formed after treatment of wild-type cells with cephalexcin to inhibit cell division, note that MinD still oscillates in striped zones; (C) are dnaXE145A cells grown in rich media for 3 h. This lack of oscillation can account for the observed division inhibition.
Figure 14: The Interaction Between MukB and ParC is Required for Stimulation of Topo IV-Catalyzed Superhelical DNA Relaxation(A) MukB stimulates wild-type Topo IV-catalyzed relaxation of negatively supercoiled DNA. However, Topo IV reconstituted from ParE and ParCR705A, a variant ParC protein that cannot interact with MukB, is not stimulated by MukB.
Figure 15: Resolution of Converging, Stalled Replication Forks by RecQ and Topo III(A) Schematic of substrate preparation and resolution. Plasmid DNA (i) is replicated in the presence of Tus, generating a late replicative intermediate (LRI, iii) where the 3'-OH ends of the nascent leading strands of the converging replication forks are separated by about 130 bp (iv). Treatment of the LRI with RecQ and Topo III yields two gapped, form II sister DNA molecules. (B) Products of the replication reaction in either the absence or presence of Tus. (C) The LRI resolution reaction. Complete resolution of the LRI to daughter form II DNA molecules requires Topo III, RecQ, and SSB.