Supplementary Figure 1. Cell long-axis histograms of the loci on the chromosome right arm. The patterns are virtually identical to the ones for the chromosome left arm in Figure 2.
Supplementary Figure 2.Radial-axis correlation of various loci. To understand the organization of the spatial relationship between the left and right chromosomes arms along the radial axis of the cell, we plotted the relative loci positions against one another. Our analysis shows three main results. (1) Loci on the same nucleoid sister but on the opposite chromosome arms tend to be widely separated along the radial axis. They occupy each cell halves along the radial axis (e.g., 54.2’-22’). (2) Loci on the same nucleoid sister and on the same chromosome arm tend to lie in the same cell half of the radial axis (e.g., 15.2’-22’). (3) Loci on different nucleoid sisters are randomly positioned with respect to each other. These trends are true for all other loci we tested. The exception is the origin-proximal loci, but this is expected because the origin spans the radial axis as a single radial-axis peak (Figure 5A and Supplementary Figure 3). Note that the cell can settle on the microscope slide at any radial angle, so that some apparent line-of-sight overlap of the arms is expected in the graph even if the two arms are completely separated.
Supplementary Figure 3.A supercomputer simulation of a ring polymer.(left panel) Simulation of a ring polymer confined in an open cylinder. The polymer is assumed to have 50 conceptual subunits. The molecular dynamics simulation of its behavior was carried out over time as described (Jung et al., 2012). The instantaneous ends (red) of the chain on average cross the radial axis and thus their radial distributions show a signature single peak, whereas the parallel arms give two peaks. This can be readily understood as both arms of the hypothetical chromosome lie away from the radial axis and would therefore both give a two-peak population distribution when radially integrated. However, as the measured position approaches the outer ends that connect the arms, the distribution flattens and eventually becomes a one-peak distribution (Figure 5). This is a direct consequence of the compressed, donut-like geometry of the nucleoid, i.e., the outer ends of the structure are loops that join the chains and usually cross the central long axis. Thus, the parallel arms of a ring polymer should give a two-peak radial-axis distribution. The distribution of the loop regions becomes increasingly flattened with sequences near the apex of the loop giving the one-peak type. Note that specific sequences affected by polymer-end proximity are expected to extend further than Supplementary Figure 3 implies (see Experimental Procedures).
Supplementary Figure 4. Partial overlap of duplicated and unduplicated foci in 2+1 and 4+2 cells. Cartoons based on the color-coded ring polymer model represent possible arrangements of the DNA blocks in typical individual 2+1 and 4+2-foci cells. The long axis orders of foci in the reference 22’(filled circles) and 92.5’ (open circles) in the 22’-92.5’population are scored on the right (ca 6,000 cells scored). These results indicate significant overlap between duplicated and unduplicated DNA within the cell during multi-fork replication.
Supplementary Movie S1.A typical timelapse movie of SJ290 strain.The movie shows two types of fluorescent fusion protein: the extended red regions for the nucleoid labeled by HU-mCherry, and the green focifor 33.7’ (terminus) marked by GFP-ParB. The two colors channels and the phase images are overlaid for each time flag. The second channel from the left is used in Figure 4B, but virtually all cells show the same sequence of events: localization of terminus at the cell center, splitting of the duplicated terminus foci, (asynchronous) movement of each terminus focus to the center of each sister nucleoid. The interval of the time-lapse is 6 minutes.
Supplementary Movie S2.Animation of spatiotemporal development of the chromosome during overlapping cell cycle using the ring polymer model.