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. 2016 Jun 2;44(10):4763-84.
doi: 10.1093/nar/gkw147. Epub 2016 Mar 6.

Diverged Composition and Regulation of the Trypanosoma Brucei Origin Recognition Complex That Mediates DNA Replication Initiation

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Free PMC article

Diverged Composition and Regulation of the Trypanosoma Brucei Origin Recognition Complex That Mediates DNA Replication Initiation

Catarina A Marques et al. Nucleic Acids Res. .
Free PMC article

Abstract

Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying.

Figures

Figure 1.
Figure 1.
Effect of induced RNAi against TbORC1/CDC6, TbORC4 or Tb3120 in PCF T. brucei. (A) Growth curves of uninduced (Tet-) and tetracycline-RNAi induced (Tet+) cell cultures over 5 (TbORC1/CDC6 and TbORC4) or 7 days (Tb3120). Cell concentration was assessed every 24 h and the mean concentration from two independent experiments is shown; error bars depict the standard error of the mean (SEM). Red arrows denote times at which a 1:10 dilution of the Tet- or Tet+ cultures was carried out at selected time points after RNAi induction. Insert box: efficiency of RNAi knockdown of mRNA levels assessed by RT-qPCR at selected time points after RNAi induction. The results represent the levels of mRNA in the Tet+ sample relative to non-induced, calculated using the ΔΔCt method. The mean of two independent experiments is shown, and error bars represent SEM. For TbORC4, the levels of the endogenously 12myc-tagged TbORC4 were assessed by western blot analysis of whole cell extracts 24 and 48 h after RNAi induction (+) or without induction (-); T.brucei Elongation factor 1 α (Ef1α) was used as a loading control. (B) Quantification of the proportion of different cell types throughout the course of the RNAi induction, based on the number of nuclei (N) and kinetoplasts (K) detected in individual cells stained with DAPI. A minimum of 150 cells were counted per time point and experimental group (Tet- and Tet+), and percentages of each cell type (1N1K, 1N2K, 2N2K, 0N1K and others) were calculated relative to the total amount of cells analysed. The graph represents the mean of each cell type observed in two independent experiments, while the error bars show SEM. (C) Percentage of cells displaying nuclear EdU signal in the Tet+ samples relative to the number of EdU positive cells in the Tet- culture from the same time point. A minimum of 150 cells were analysed per time point and group (Tet- and Tet+) and the mean from two independent experiments is shown; error bars represent the SEM. The insert in the Tb3120 column shows an example of an EdU positive cell, relative to the same cell DAPI-stained and DIC imaged; scale bar represents 5 μm.
Figure 2.
Figure 2.
Effect of induced RNAi against TbORC1B in PCF T. brucei. (A) Growth curves of uninduced (Tet-) and tetracycline-RNAi induced (Tet+) cell cultures over five days, where cell concentration was assessed every 24 h; mean concentration from two independent experiments is shown and error bars depict the standard error of the mean (SEM). Insert box: efficiency of RNAi knockdown of TbORC1B mRNA levels assessed by RT-qPCR. The results represent the amount of mRNA at the time points shown after RNAi induction (Tet+) relative to the non-induced sample (Tet-). The mean of two independent experiments is shown, and the error bars represent SEM. (B) Quantification of the proportion of different cell cycle types throughout the course of the RNAi induction, based on the number of nuclei (N) and kinetoplasts (K) detected in individual cells stained with DAPI. A minimum of 150 cells were counted per time point and experimental group (Tet- and Tet+), and percentages of each cell type (1N1K, 1N2K, 2N2K, 0N1K and others) were calculated relative to the total amount of cells analysed. The graph represents the mean of each cell type observed in two independent experiments, while the error bars show SEM. (C) Percentage of cells displaying nuclear EdU signal in the Tet+ samples relative to the number of EdU positive cells in the Tet- culture from the same time point. A minimum of 150 cells were analysed per time point and group (Tet- and Tet+) and the mean from two independent experiments is shown; error bars represent the SEM. (D) Histograms representing the distribution of the cell population according to DNA content (stained with PI) assessed by flow cytometry at the 6, 12, 24 and 48 h time points. Approximately 30 000 cells were analysed per sample, and the histograms represent the percentage of cells in the population, normalized to mode; cells with 2n and 4n DNA content are indicated.
Figure 3.
Figure 3.
Immunofluorescence localization of TbORC1/CDC6, TbORC4, Tb3120 and Tb7980 in PCF T. brucei cells. Analysis of the subcellular localization of 12myc tagged variants of TbORC1/CDC6 (A), TbORC4 (B), Tb3120 (C) and Tb7980 (D) are shown in fixed cells. In each case the cells are shown stained with DAPI, allowing their classification as 1N1K cells (G1 phase), 1N1eK cells (S phase), 1N2K cells (G2/M phase) and 2N2K cells (post-mitosis). The tagged proteins are detected in each cell type using an anti-myc antiserum coupled with the Alexa Fluor 488 flurophore (myc, green). Finally, the outline of all cells is shown by DIC imaging. Arrows direct the reader's attention to single cells of the expected cell cycle stage if more than one cell is shown. Images were acquired using a DeltaVision imaging system and deconvolved using the ratio conservative method, on SoftWoRx software. The scale bar represents 5 μm.
Figure 4.
Figure 4.
Cell-cycle dependent nuclear localization of TbORC1B. (A) Percentage of cells containing nuclear myc signal in untagged T. brucei PCF cells (927 wt) relative to PCF cells expressing TbORC1/CDC6–12myc, TbORC1B-12myc, TbORC4–12myc, 12myc-Tb7980 or Tb3120–12myc, each from the endogenous locus; the mean of three independent experiments is shown (>125 cells each) and error bars show SEM. (B) Immunofluorescent detection of TbORC1B-12myc with anti-myc antiserum (middle row) in 1N1K cells (G1 phase), 1N1eK cells (S phase), 1N2K cells (G2/M phase), and 2N2K cells (post-mitosis), stained with DAPI (top row) and cell outline shown by DIC (lower row). Arrows highlight a single cell of the expected cell cycle stage if more than one cell is captured in the images. The scale bar represents 5 μm. (C) The proportion of cell cycle stages displaying TbORC1B-12myc signal is shown either as the percentage of total cells (insert box), or as the percentage of positive cells (main graph), as determined by N-K ratio (dark blue, 1N1K cells; light blue, 1N1eK cells; yellow, 1N2K cells, red, 2N2K). The mean is shown from two independent experiments, and the error bars depict standard deviation. (D) Percentage of individual cell cycle stage cells that display TbORC1B-12myc signal (as no 2N2K cells were detected to have TbORC1B-12myc signal, these are not represented); the mean of four independent experiments is represented and error bars show standard deviation. Statistical significance between the different groups was assessed using the one-way ANOVA parametric test: (***) P-value < 0.001; (****) P-value <0.0001. (E) Asynchronous TbORC1B-12myc expressing cells were separated into G1, Early S, Late S and G2/M phases FACS sorting and the western blot shows the sorted fractions probed with anti-myc antiserum (α-myc); the same blot was probed with antiserum against the transcription elongation factor Ef1α as a loading control.
Figure 5.
Figure 5.
Quantification of TbORC1/CDC6 and TbORC1B subcellular localization through the cell cycle. Intensity of the DAPI (left graphs, blue dots) and myc signals (right graphs, green dots) of PCF T. brucei cells expressing TbORC1/CDC6–12myc (A) or TbORC1B-12myc (B) is represented as the mean pixel intensity within a circular ROI (21 × 21 pixels), drawn around each individual cell nucleus; for the myc signal data, the red dotted line represents the average background signal measured in 927 wt cells that do not express any tagged protein. Dark green dots represent cells in which TbORC1B-12myc signal was not visually detected, while light green dots represent cells with visually detectable TbORC1B-12myc signal. In each case, cells are separated by cell cycle stage, determined by N-K ratio in the DAPI images: 1N1K cells (G1 phase), 1N1eK cells (S phase), 1N2K cells (G2/M phase) and 2N2K cells (post-mitosis). The median values derived from the analysis of 591 TbORC1/CDC-12myc cells (1N1K – 1.47; 1N1eK – 2.31; 1N2K – 2.86; 2N2K – 1.48) and 412 TbORC1B-12myc cells (1N1K – 0.48; 1N1eK – 1.99; 1N2K – 0.71; 2N2K – 0.48) are represented, with error bars depicting the interquartile range. Statistical significance between the different cell cycle stages was assessed using the Kruskal-Wallis non-parametric test: (*) P-value < 0.05; (**) P-value < 0.01; (****) P-value < 0.0001.
Figure 6.
Figure 6.
Super-resolution imaging of TbORC1/CDC6, TbORC4, TbORC1B and TbMCM3 through the cell cycle. PCF T. brucei cells expressing TbORC1/CDC6–12myc, TbORC4–12myc, TbORC1B-12myc or TbMCM3–12myc were incubated for 3 h with 150 μm EdU, fixed and stained with DAPI and with AlexaFluor® 488-conjugated anti-myc antibody, while EdU was detected with AlexaFluor® 594-conjugated azide. Images were acquired with a Zeiss Elyra super-resolution microscope system in SIM mode. In each case, representative maximum projection images are shown of cells in the different cell cycle stages, determined by N-K ratio in the DAPI images: 1N1K cells (G1 phase), 1N1eK cells (S phase), 1N2K cells (G2/M phase) and 2N2K cells (post-mitosis). All cells are shown as a merge of the EdU (red), anti-myc antiserum (green) and DAPI (blue) signals, whose intensities are quantified in the plots to the left; signal intensities (y-axes, arbitrary units) were analysed in a horizontal line across the boxed area surrounding the nucleus.
Figure 7.
Figure 7.
Effect of induced RNAi against TbORC1/CDC6 or TbORC1B in bloodstream form T. brucei. (A) Growth curves of uninduced (Tet-) and tetracycline-RNAi induced (Tet+) cells targeting TbORC1/CDC6 (left) or TbORC1B (right) are shown over 3 days. Cell concentration was assessed every 24 h and the mean concentration from two independent experiments is shown; error bars depict the SEM. Levels of 12myc-tagged protein are shown at 24 h time with (+) or without (-) RNAi induction by western blotting of whole cell extracts with anti-myc antiserum (α-myc); loading controls are shown by probing the same blot with antiserum against T.brucei Ef1α; size markers are indicated. (B) Quantification of the proportion of different cell types throughout the course of the RNAi induction, based on the number of nuclei (N) and kinetoplasts (K) detected in individual cells stained with DAPI. A minimum of 125 cells were counted per time point and experimental group (Tet- and Tet+), and percentages of each cell type (1N1K, 1N2K, 2N2K, 0N1K and others) were calculated relative to the total amount of cells analysed. The graph represents the mean of each cell type observed in two independent experiments, while the error bars show SEM. (C) Percentage of cells displaying nuclear EdU signal in the Tet+ samples relative to the number of EdU positive cells in the Tet- culture from the same time point. A minimum of 125 cells were analysed per time point and group (Tet- and Tet+) and the mean from two independent experiments is shown; error bars represent the SEM. (D) Examples of aberrant cells after TbORC1/CDC6 RNAi, stained with DAPI, and cell outline shown as a DIC image.
Figure 8.
Figure 8.
Gel filtration of T. brucei cell extracts. (A) Detection of TbORC1/CDC6–12myc with the myc antiserum in the different fractions resulting from gel filtration of TbORC1/CDC6 -/12myc cell line lysates. Multiple membranes spanning the fractions are aligned, and the estimated molecular weight is shown next to the corresponding fraction by a black arrow. The eluted volume (ml) corresponding to each fraction is depicted below each membrane. (*) pinpoints the lanes loaded with the lysed sample prior gel filtration, as a positive control in each western blot membrane. Size markers (kDa) are indicated. (B) Gell filtration of cell extracts from T. brucei PCF cells expressing either TbORC1/CDC6–12myc and TbORC4–6HA, or TbORC1/CDC6–12myc and TbMCM3–6HA. Extracts from the two cell lines were combined prior to gel filtration and proteins were detected with anti-myc and anti-HA antisera; expected sizes of the epitope tagged proteins are indicated.
Figure 9.
Figure 9.
Potential ORC architecture in T. brucei. Architecture of the D. melanogaster origin recognition complex (ORC; composed of Orc subunits numbered 1–6), which interacts (arrow) with the Orc1-related factor Cdc6, is shown based on the structure determined by (22). In T. brucei, recognisable ORC subunit orthologues of Orc1 and Orc4 (TbORC1/CDC6 and TbORC4, respectively) are identified using the same colours and solid outlines, while putative orthologues of Orc2 and Orc5 (Tb3120 and Tb7980, respectively) are shown by dotted circles and lighter colours; subunits that are absent or highly diverged are shown by unfilled dotted circles. TbORC1B interacts with TbORC1/CDC6, but appears to not be a static ORC component, and hence its inclusion in ORC is uncertain.

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