DNA Replication-Dependent Formation of Joint DNA Molecules in Physarum polycephalum  Marianne Bénard, Chrystelle Maric, Gérard Pierron  Molecular Cell  Volume 7, Issue 5, Pages 971-980 (May 2001) DOI: 10.1016/S1097-2765(01)00237-4

Figure 1 Kinetics of DNA Replication at the proP Locus of Physarum Using Synchronous DNA Samples The structure of the 5 kb EcoRI fragment containing the proP gene and its replication origin is depicted. Introns appear as white segments and arrows indicate the polarity of transcription and bidirectionality of replication. Below, a scheme represents the mitotic cycle in the plasmodium. There is no G1 phase. Typical duration of mitosis (M) and S and G2 phases is given in hours. Descending arrows indicate the cell cycle stages at which DNA samples were obtained for 2D gel analysis. Above each frame, the time in minutes with respect to the onset of S phase is given. Below each 2D gel, a scheme represents the DNA structures detected by hybridization. At stages where X-shaped DNA molecules are observed (arrows within the frames), the percentage of DNA molecules with X-shaped and Y structures were quantified by phosphorimaging and are reported Molecular Cell 2001 7, 971-980DOI: (10.1016/S1097-2765(01)00237-4)

Figure 2 Joint DNA Molecule Formation and Temporal Order of Replication at Two Unlinked Loci, Using a Single Crosshybridizing Probe (A) Schematic representation of the four EcoRI fragments recognized by the DNA probe derived from the ardC promoter. On the left, the 6.6 and 7.2 kb ardC1 and ardC2 allelic fragments are represented. The position of the gene, its replication origin, and the 0.7 kb DNA sequence found only in the ardC2 allele are shown. The probe crosshybridizes with an anonymous locus for which the two alleles S1 and S2 (for spot 1 and spot 2) are polymorphic as 3.0 and 4.0 kb EcoRI fragments. (B) Kinetics of replication and X-DNA formation over these two unlinked loci. Each frame represents a different cell cycle time point indicated on top. In mitosis, only linear DNA molecules are detected. At +5 min in S phase, bubbles and Y replication intermediates (B/Y) demonstrate active replication of the ardC1 and ardC2 alleles. In contrast, the S2 crosshybridizing fragment is not replicating (the S1 spot is not visible on this blot). At +20 min in S phase, joint DNA molecules are seen over the almost completely replicated actin ardC gene alleles (XC1 and XC2) and are not seen in the S1 and S2 fragments that are now engaged in replication. These two fragments are passively replicated as indicated by the Y arcs (YS1 and YS2). At +40 min in S phase, both loci are fully replicated and both contain joint DNA molecules Molecular Cell 2001 7, 971-980DOI: (10.1016/S1097-2765(01)00237-4)

Figure 3 Comparison of the Frequency of Joint DNA Molecules Detected within Two Unlinked Replicons A single blot of EcoRI-digested DNA extracted at +25 min in S phase was hybridized with the 1.1 kb ardC gene probe and reprobed with the proP gene probe. Both genes are replicated at the onset of S phase from promoter proximal, bidirectional replication origins (see schematic representation). The downstream EcoRI site of the ardC gene locus is polymorphic (EcoRI*). Following hybridization, joint DNA molecules were quantified by phosphorimaging Molecular Cell 2001 7, 971-980DOI: (10.1016/S1097-2765(01)00237-4)

Figure 4 Distribution of the Junctions within Adjacent and Overlapping Fragments of the Actin ardC Gene Locus The structure of the locus is drawn. EcoRI and HindIII restriction patterns detected by hybridization with the upstream (a) or downstream (b) probes are shown. A single blot of HindIII-digested DNA was used to compare directly the frequency of the joint DNA molecules by consecutive hybridizations with the (a) and (b) probes in the blots shown on top. Probe (a) was also used to analyze the EcoRI digest of the same DNA preparations in blots shown on the bottom. The downstream HindIII and EcoRI sites are polymorphic (*) so that allelic fragments are separated by 2D gel. Phosphorimaging quantitation reveals a similar variation in the distribution of the X-shaped DNA molecules, expressed as a percent of total radioactivity within the locus at +25 min (on the left) and +40 min (on the right), even though fewer X-shaped molecules are seen at +40 min Molecular Cell 2001 7, 971-980DOI: (10.1016/S1097-2765(01)00237-4)

Figure 5 Joint DNA Molecules Are Labile under Conditions that Promote Branch Migration (A) Resolution of the junctions by heat treatment. Two identical HindIII-digested samples of a synchronous, early S phase (+25 min) DNA preparation were analyzed by 2D gel electrophoresis. After the first dimension, one agarose lane was incubated at 65°C for 4 hr in branch migration buffer, and the other was kept in the electrophoresis buffer at room temperature as a control. Both lanes were then electrophoresed in a second dimension, and blotted and hybridized with probe (a) to detect the 6.6 kb fragment upstream of the actin ardC gene (see Figure 4). The percentages of linear (L)-, Y-, and X-shaped hybridizing structures were measured in the control and heat-treated samples. (B) Magnesium reduces the ability of the joint DNA molecules to branch migrate. The stability of joint DNA molecules during incubation at 65°C for 2 hr in the absence (left) or the presence (right) of 10 mM magnesium was determined as in (A). The percentages of joint DNA molecules resistant (within the spike) and resolved during the incubation at 65°C (spot of linear molecules under the spike) were measured by phosphorimaging Molecular Cell 2001 7, 971-980DOI: (10.1016/S1097-2765(01)00237-4)

Figure 6 Joint DNA Molecules Are Formed between Sister Chromatids Schematic representation of the joint DNA molecules formed either between the newly synthesized sister chromatids (on top) or between homologous chromosomes (below) at a site showing a polymorphism of restriction sites. Results obtained with HindIII-digested genomic DNA extracted 25 min after the onset of S phase are shown on the bottom. Polymorphic restriction fragments of 4.8 and 5.2 kb are detected with probe (b) of the 3′ end of the ardC gene (see Figure 4). A spike of X-shaped DNA molecules is observed for each of the two alleles, as predicted for joint DNA molecules associating sister chromatids Molecular Cell 2001 7, 971-980DOI: (10.1016/S1097-2765(01)00237-4)

Figure 7 No Joint DNA Molecules Are Formed upon Inhibition of DNA Replication A single plasmodium was cut into two parts with scissors 20 min before mitosis, a stage characterized by the rapid migration of the nucleolus toward the nuclear envelope as shown in the scheme. Half of the plasmodium was treated with 50 mM hydroxyurea (+HU) to inhibit DNA replication. HindIII-digested DNA from the control (−HU) and treated halves (+HU) was analyzed by 2D gel and probed with probe (a) of the actin ardC gene. Below each frame, the structure of the nonlinear DNA molecules detected is represented by a scheme. DNA replication of the HindIII fragment is completed at this stage, with forks (Y arc) in less than 1% of the copies of the fragment. As expected, joint DNA molecules (X) are present in the control sample. In contrast, no joint DNA molecules are formed upon inhibition of DNA replication by hydroxyurea. A block in DNA replication is evidenced by the accumulation of replication intermediates at the two sites Y1 and Y2 (see Results) Molecular Cell 2001 7, 971-980DOI: (10.1016/S1097-2765(01)00237-4)