Fig 1.
Schematics of experimental setup.
A: Schematics of timecourses using single EdU pulse labels. B: Schematic of experiments using sequential pulses of two nucleotides (EdU and BrdU) to determine the length of each replicative round. C: Schematics of timecourses using 2-nucleotide (EdU and BrdU) interval pulse labels.
Fig 2.
Length and dynamics of S-phase in P. falciparum and P. knowlesi.
A, B: Scatter graphs of nuclear numbers at each hour in P. falciparum (A) or P. knowlesi (B), n = 100 cells, medians are shown in red. Biological replicate experiments are shown in S3 Fig. C, D: Percentage of 100 cells showing some EdU labelling at each hour in P. falciparum (C) and P. knowlesi (D). E, F: Percentage of replicating nuclear masses per cell at each timepoint in P. falciparum (E) and P. knowlesi (F), n = 100, medians are shown in red. G, H: Percentage of replicating nuclear masses per cell, re-plotted by number of such masses, in P. falciparum (G) and P. knowlesi (H), n = 100, medians are shown in red.
Fig 3.
Patterns of DNA replication within nuclei in P. falciparum and P. knowlesi.
A: Schematic of the timecourses shown in this figure. B: Examples of 5 distinct patterns of EdU incorporation seen in replicating nuclei (P. falciparum are shown, representative of patterns in both species; dotted line highlights the relevant nuclear mass). Scale bar all panels 1μm. C: Representative examples of cells at each hour across the timecourse for P. falciparum and P. knowlesi. 46-47hpi in P. falciparum and 31-32hpi in P. knowlesi show segmented schizonts; 33hpi in P. knowlesi shows a reinvaded ring. Scale bar all panels 2μm. D: Percentages of each distinct pattern seen at each point across the timecourse in P. falciparum and P. knowlesi. E: Intensity of EdU staining (calculated as ‘integrated density’ across each nuclear mass, i.e. the sum of values for fluorescent signal in all pixels in each mass) seen at each timepoint in P. falciparum and P. knowlesi. A significant drop in EdU staining intensity is seen at the end of the timecourse in both species (***, p = 0.001, ANOVA). In P. falciparum, this correlates clearly with the rise at 44-47hpi in the pattern qualitatively categorised as ‘less bright’ in (C). Intensities in nuclear masses with each pattern of staining are shown separately in S4 Fig. Nuclei that had entirely finished replicating and showed no detectable signal were excluded.
Fig 4.
Replicative rounds become faster across the course of schizogony.
A: Schematic of the timecourses shown in this figure. (Biological replicate experiments are shown in S7 Fig). B: Representative examples of P. falciparum cells labelled with EdU for 30 minutes and then a 15-minute pulse of BrdU at increasing intervals. Scale bar all panels 2μm. C: Graphs showing the percentage of all nuclear masses that labelled with EdU and BrdU, firstly in mono-nuclear cells and then for 2-n, 3-6n and >6-n cells (n = 20). Scatter plots display the same data broken down per-cell, with means shown for each dataset. D: Representative examples of P. knowlesi cells labelled with EdU for 30 minutes and then a 15-minute pulse of BrdU at increasing intervals. Scale bar all panels 2μm. E: Graphs as in (C) showing the percentage of nuclear masses labelled with EdU and BrdU in P. knowlesi cells of increasing ploidy (n = 20 per category).
Fig 5.
Gap phases lengthen across the course of schizogony.
A: Schematic of the timecourses shown in this figure. B,D: Representative examples of cells across double-labelled timecourses for P. falciparum (B) and P. knowlesi (D). P. falciparum was labelled at 3h intervals, up a maximum of 9h (35-44hpi); P. knowlesi at 2h intervals, up a maximum of 10h (18-28hpi). Scale bars all panels 2μm. C,E: Percentages of nuclear masses per cell labelled with EdU and BrdU, EdU alone, or BrdU alone throughout each timecourse. Dotted lines separate the datasets from different time-intervals. Medians are shown in red.
Fig 6.
Long-term arrest of individual nuclei is common during schizogony.
A: Representative examples of P. falciparum cells double-labelled with EdU for 30 minutes and then BrdU for 5h. Scale bar all panels 2μm. B: Percentages of all nuclear masses labelled with EdU and BrdU, EdU alone, BrdU alone, or neither label (n = 20 cells, stratified into cells with up to 6 nuclear masses or more than 6). C: Scatter plots showing percentages of nuclear masses per cell with the various labelling patterns. Medians are shown for each dataset. D: Representative examples of P. knowlesi cells double-labelled with EdU for 30 minutes and then BrdU for 5h. Scale bar all panels 2μm. E: Percentages of all nuclear masses labelled with EdU and BrdU, EdU alone, BrdU alone, or neither label (n = 20 cells, stratified into cells with up to 6 nuclear masses or more than 6). F: Scatter plots showing percentages of nuclear masses per cell with the various labelling patterns. Medians are shown for each dataset.
Fig 7.
Single-molecule DNA replication dynamics in P. falciparum and P. knowlesi.
A: Schematic showing possible patterns of labelling on DNA fibres. Pattern 1 is an ongoing replication fork, moving throughout the two consecutive pulse-labels. Pattern 2 shows origins that fired during the first and second pulse labels, with inter-origin distance (IOD) measured as centre to centre distance between two adjacent origins. Pattern 3 shows two replication forks terminating during the first or second pulses. The presumed positions of the replication origins are indicated with asterisks in the middle of bidirectional replication forks. Arrows represent the direction of the replication forks’ progression. B: Representative DNA fibre spreads from P. falciparum and P. knowlesi. A 5kb scale bars is indicated. C: Dot plot showing distribution of replication fork speeds in P. falciparum and P. knowlesi, calculated on the basis of a 20-min pulse label (10 mins EdU, 10mins BrdU) and a stretching factor of 2.59kb/μm. Red bars represent the mean value. D: Dot plot showing distribution of inter-origin distances (IODs) in P. falciparum and P. knowlesi, calculated on the basis of a stretching factor of 2.59kb/μm. Red bars represent the mean value.