DNA replication protein Cdc45 directly interacts with PCNA via its PIP box in Leishmania donovani and the Cdc45 PIP box is essential for cell survival

Abstract

DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in Leishmania donovani Cdc45. This motif is typically linked to interaction with the eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can potentially bind upto three different proteins simultaneously via a loop region present in each monomer. Multiple binding partners have been identified from among the replication machinery in other eukaryotes, and the concerted /sequential binding of these partners are central to the fidelity of the replication process. Though conserved in Cdc45 across Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such an interaction in the in vivo context. Having confirmed the importance of Cdc45 in Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole cell extracts, also interacting with each other directly in vitro. The interaction is mediated via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to be essential for cell survival here as well. Our results implicate a role for the Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-PCNA interaction might help tether PCNA and associated replicative DNA polymerase to the DNA template, thus facilitating replication fork elongation. Though multiple replication proteins that associate with PCNA have been identified in other eukaryotes, this is the first report demonstrating a direct interaction between Cdc45 and PCNA, and while our analysis suggests the interaction may not occur in human cells, it indicates that it may not be confined to trypanosomatids.

Fig 1. Depletion of Cdc45 causes growth and cell cycle aberrations.

a. Analysis of cdc45 expression in genomic knockout lines using real time PCR analysis. Tubulin served as internal control. cdc45^-/+/+ refers to cdc45^-/+/+::hyg cells. The average of data from three experiments is presented here. Error bars represent standard deviation. Two-tailed student’s t-test was applied: *** p < 0.0005. b. Analysis of growth of Cdc45-depleted cells. Growth was initiated at 1x10⁶ cells/ml, from stationary phase cells. Left panel: single allele replacement lines compared to control cells. Ld1S::hyg and Ld1S::neo are control Ld1S cells harboring the hyg^r and neo^r genes respectively, previously found to grow at rates comparable to wild type Ld1S cells. Right panel: double allele and single allele replacement lines compared to control cells. Ld1S::neo-hyg are control Ld1S cells expressing both neo^r and hyg^r casettes, previously found to grow at rates comparable to wild type Ld1S cells. In each case three experiments were set up in parallel and the graphs represent average values, with error bars representing standard deviation. c. Determination of generation time of double allele replacement cells cdc45^-/-/+ in comparison with control Ld1S::neo-hyg cells. Growth initiated at 1x10⁶ cells/ml from exponentially growing promastigotes, and cells diluted to 1x10⁶ cells/ml every 24 hours. Average of three experiments are plotted, and error bars depict standard deviation. d. Flow cytometry analysis of Cdc45-depleted cells in comparison with control cells. Cells were synchronized at G1/S by HU treatment and then released into S phase. Time-points at which cells were sampled are indicated above each column of histograms e.g. 3h R signifies 3 hours after release from HU. M1, M2 and M3 gates represent cells in G1, S and G2/M respectively. 30000 events were counted in each case. cdc45^+/+/+—Ld1S::neo-hyg cells. cdc45^-/+/+—cdc45^-/+/+:: hyg (heterozygous) knockout cells. cdc45^-/-/+—cdc45 double allele knockout cells. The experiment was done thrice, and one data set is presented here. The percentage of gated cells in each of the cell cycle stages (G1, S and G2/M) from all three experiments are tabulated in S1 Table. e. HU-treated cells were released into S phase and 1 ml aliquots pulsed with EdU for 15 min. Left panel: Pulsing with EdU 3 hours, 4.5 hours, 6.5 hours and 10 hours after release. Right panel: Pulsing logarithmically growing cells with EdU. cdc45^+/+/+—Ld1S::neo-hyg cells. Data presented in bar chart are average of three experiments and error bars represent standard deviation. Two-tailed student’s t-test was applied: **p < 0.005, ***p < 0.0005.

Fig 2. Ectopic expression of Cdc45 in cdc45^-/-/+ cells allows the cells to overcome defects associated with Cdc45 depletion.

a. Western blot analysis of cell extracts using anti-FLAG antibody (Sigma, 1:1000 dil). cdc45^-/-/+/vector- cdc45^-/-/+ cells harboring pXG-FLAG(bleo) vector. cdc45^-/-/+::Cdc45-FL—cdc45^-/-/+ cells harboring pXG-Cdc45-FLAG(bleo) i.e. the rescue line. Loading control: tubulin. b. Left panel: Analysis of growth pattern of rescue line cdc45^-/-/+::Cdc45-FL in comparison with cdc45^-/-/+cells and control cells (Ld1S::neo-hyg-bleo—Ld1S::neo-hyg cells harboring pXG-FLAG (bleo) empty vector). Right panel: Analysis of growth pattern of line cdc45^-/-/-::Cdc45-FL in comparison with cdc45^-/-/+::Cdc45-FL cells and cdc45^-/-/+cells. Growth was initiated at 1x10⁶ cells/ml from stationary phase cells. Graphs represent average values of three experiments, with error bars representing standard deviation. c. Flow cytometry analysis of rescue line cdc45^-/-/+::Cdc45-FL in comparison with cdc45^-/-/+cells and cdc45^+/+/+ control cells (Ld1S::neo-hyg-bleo cells). 30000 events counted in every sampling. Sampling times indicated above the histograms. Cells representing G1, S and G2/M phases indicated by gates M1, M2, M3. Experiment done thrice, one data set depicted here. d. Analysis of cell survival within host macrophages. cdc45^+/+/+ control cells—Ld1S::neo-hyg-bleo cells. Intracellular parasites were scored by Z-stack imaging of DAPI-stained infected macrophages using confocal microscopy. Bar chart depicts averages of three experiments; error bars indicate standard deviation. Two-tailed student’s t-test was applied: **p<0.005; ***p < 0.0005; ns-not significant. e. Left panel: Western blot analysis of extracts isolated from promastigotes (P) and intracellular parasites (I.P.) expressing Cdc45-FLAG. As the Leishmania PCNA antibody does not cross-react with mouse PCNA (which is also of lower molecular weight) it was used as the loading control to ensure comparable loading of parasite total protein. The experiment was done thrice. Right panel: quantification of Cdc45 using Leishmania PCNA as internal control, using ImageJ software. Average of three experiments is shown, with error bars indicating standard deviation. Student’s t-test was applied: *p<0.05.

Fig 3. Structural analysis of Leishmania donovani Cdc45.

a. Sequence alignment of Leishmania donovani Cdc45 against human Cdc45 generated by Phyre2. Based on structural analysis of human Cdc45 by Simon et al [9], α10-α14 are helices that form the Mcm2/5 binding face. All other marked helices and β strands represent regions involved in interaction with GINS b. Upper left panel: Human Cdc45 ribbon representation derived from crystal structure (PDB ID: 5DGO) as reported by Simon et al [9]. Navy blue regions: Complex Interaction Domain (CID) that interfaces with Mcm2/5. Green regions: Domains that interface with GINS complex. Red region: PIP box. Sequence of PIP box indicated below structure. Upper right panel: Ribbon representation (magenta) of 3D model of Leishmania donovani Cdc45 modelled using Phyre2 against human Cdc45 (PDB ID: 5DGO) as template. Navy blue region indicates α12 helix. Black arrowheads and associated numbers indicate amino acid stretches that have been excluded by Phyre2 during modeling. Red arrowhead and associated numbers indicates location of PIP box (also excluded during modeling). Sequence of PIP box indicated below structure. Lower panel: View of superimposed structures of LdCdc45 and 5DGO, using PyMOL. Though showing overall structural similarity (RMSD value 2.2Å), the PIP motifs of the two structures do not overlap.

Fig 4. LdCdc45 interacts with PCNA via its PIP box.

a and c. Western blot analysis of PCNA immunoprecipitates from lysates of cells expressing a. Cdc45-FLAG ectopically (indicated as Cdc45-FLAG in the figure) c. Cdc45-FLAG or Cdc45-PIP-FLAG ectopically (indicated as Cdc45-FLAG or Cdc45-PIP-FLAG in the figure). Ld1S+vector was used as control (Ld1S cells harboring pXG-FLAG vector which carried the neo^r drug cassette). IP: immunoprecipitation. IB: immunoblotting (western blot). Immunoblots were probed with anti-FLAG (Sigma, 1:1000 dil), anti-PCNA (raised earlier in the lab, 1:2500 dil) and anti-IgG (Jackson ImmunoResearch, 1:10000 dil) antibodies. Both experiments were carried out twice with similar results; one data set of each is shown. b. and d. Analysis of pull down reactions using b. PCNA and MBP-Cdc45_481-785 d. PCNA and MBP-Cdc45_481-785 or PCNA and MBP-Cdc45-PIP_481-785, by Coomassie staining and western blotting. IB: Immunoblotting. Immunoblots were probed with anti-MBP (Sigma, 1:12000 dil) and anti-PCNA (1:2500 dil) antibodies. Both experiments were done twice with comparable results; one data set of each is shown. e. Analysis of purified MBP-Cdc45_481-785 proteins by Coomassie staining of SDS-PAGE (left panel) and western blotting using anti-MBP antibodies (right panel). f. Analysis of pull down reactions using MBP and PCNA, by Coomassie staining and western blotting. g. Analysis of pull down reactions using MBP-Cdc45_481-785 proteins (wild type and PIP mutant) and PCNA, by Coomassie staining and western blotting. Both experiments were performed twice with comparable results; one data set of each is shown.

Fig 5. Cdc45-PIP box is essential for Leishmania cell survival.

a. Western blot analysis of whole cell lysates isolated from transfectant cdc45^-/-/+ promastigotes expressing Cdc45-FLAG or Cdc45-PIP-FLAG. b. Analysis of growth patterns of transfectant cdc45^-/-/+ promastigotes expressing Cdc45-FLAG or Cdc45-PIP-FLAG. Graph presents averages of three experiments with error bars indicating standard deviation. c. Flow cytometry analysis of transfectant cdc45^-/-/+ promastigotes expressing Cdc45-FLAG or Cdc45-PIP-FLAG. The experiment was performed thrice, and the data from one experiment is presented here. 30000 events were analyzed at every sampling. Sampling times are indicated along the y axes and cell types are indicated below the histogram columns. cdc45^+/+/+—Ld1S::neo-hyg-bleo cells.

Fig 6. Cdc45 helps recruit/stabilize PCNA-polymerase complexes on chromatin during active DNA replication.

a-e. Analysis of soluble and chromatin-bound protein fractions isolated from cdc45^-/-/+::Cdc45 and cdc45^-/-/+::Cdc45-PIP cells. Each experiment was carried out thrice. One data set for each is presented here. Reactions resolved on 12% SDS-PAGE were probed with various antibodies as indicated. S1, S2 –soluble fractions; S3, S4 –DNA-associated fractions. Mr- molecular weight marker a. Outline of fractionation scheme b. Isolation from 5 x10⁷ logarithmically growing cells. c. Isolation from 5 x10⁷ HU treated cells d. Isolation from 1–2 x10⁹ synchronized cells 2 hours after release from block. e. ratio of chromatin-bound PCNA (S3+S4): histone H4 (S3+S4) in cdc45^-/-/+::Cdc45-PIP cells, was plotted as a percentage of ratio of chromatin-bound PCNA (S3+S4): histone H4 (S3+S4) in cdc45^-/-/+::Cdc45 cells. Quantification was carried out using ImageJ software. Bars represent average of three experiments, with error bars indicating standard deviation. Student's t-test was applied for statistical significance. p value <0.05*, <0.005**, <0.0005*** f. ratio of chromatin-bound Mcm4 (S3+S4): histone H4 (S3+S4) in cdc45^-/-/+::Cdc45-PIP cells, was plotted as a percentage of ratio of chromatin-bound Mcm4 (S3+S4): histone H4 (S3+S4) in cdc45^-/-/+::Cdc45 cells. Similar analysis was also done for chromatin-bound Cdc45-FLAG. Quantification was carried out using ImageJ software. Bars represent average of three experiments, with error bars indicating standard error of mean. Student's t-test was applied for statistical significance. ns: not significant. g-h. Analysis of PCNA immunoprecipitates from whole cell extracts of cdc45^-/-/+::Cdc45 and cdc45^-/-/+::Cdc45-PIP cells. g. Cells were treated with 5 mM HU for 8 hours, with 20 μM MG132 being added 5 hours into the treatment. h. Cells were harvested 2 hours after release from HU-induced block. 20 μM MG132 was added 5 hours into the HU treatment and also added to the cells at the time of release from HU. The immunoprecipitates were resolved on SDS-PAGE and analysed for ubiquitinated PCNA using anti-Ub antibodies (Santa Cruz, 1:1000 dil). The blot was subsequently probed with anti-PCNA antibody (1:2500 dil).

Fig 7. Cdc45-PIP box is essential for S. pombe cell survival.

a. Upper panel: Sequence alignment of Schizosaccharomyces pombe Cdc45 against human Cdc45 generated by Phyre2. PIP boxes highlighted in red. Lower left panel: Human Cdc45 ribbon representation derived from crystal structure (PDB ID: 5DGO; [9]). Navy blue region indicates α12 helix, red region depicts PIP box, sequence of PIP box indicated below structure. Lower right panel: Ribbon representation (pink) of 3D model of Schizosaccharomyces pombe Cdc45 modelled with Phyre2 using human Cdc45 (PDB ID: 5DGO) as template. Black arrowheads and associated numbers indicate amino acid stretches that have been excluded by Phyre2 during modeling. Navy blue region indicates α12 helix. Red region indicates PIP box. Sequence of PIP box indicated below structure. b. Western blot analysis of whole cell lysates isolated from sna1^goa1 transformants expressing LdCdc45 (wild type or PIP mutant) proteins. Two clones of each type were analyzed, using anti-FLAG antibodies (1:1000 dil). c. Complementation assays. sna1^goa1 transformants expressing LdCdc45 (wild type or PIP mutant) proteins were streaked on EMM2 plates (with necessary supplements) and incubated at permissive and non-permissive temperatures to assess functional complementation. Transformant expressing SpCdc45 served as positive control for complementation. Both clones of each transformant were streaked out on the same plate, one on each half. The experiment was done twice with comparable results; one set is presented here. d. Western blot analysis of whole cell lysates isolated from sna1^goa1 transformants expressing SpCdc45 (non-tagged wild type or His-tagged PIP mutant) proteins. Two clones of SpCdc45-PIP were analyzed, using anti-His antibodies (1:10000 dil). e. Complementation assays. sna1^goa1 transformants expressing SpCdc45 (wild type or PIP mutant) proteins were streaked on EMM2 plates and incubated at permissive and non-permissive temperatures. Both clones were streaked out on the same plate, one on each half. The experiment was done twice with comparable results; one set is presented here.

Fig 8. The role of Cdc45-PCNA interaction at the replication fork.

The active replisome on the leading strand template includes several proteins such as the Cdc45-MCM-GINS complex, DNA pol ε, and PCNA. Here, the heterotetrameric GINS is represented as a single entity. DNA pol ε is depicted with only two subunits: PolE1 (bi-lobed) and PolE2, as the other two subunits have not been identified in trypanosomatids. No Ctf4 has yet been identified in trypanosomatids. The interaction of Cdc45 with PCNA, the sliding clamp processivity factor of the replicative polymerases, may further stabilize the polymerase on the advancing replisome machinery, thus enhancing the efficiency of replication.