Ciardo D et al. (2021), Polo-like kinase 1 (Plk1) regulates DNA replica...

XB-ART-58412

Nucleic Acids Res 2021 Sep 27;4917:9851-9869. doi: 10.1093/nar/gkab756.

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Polo-like kinase 1 (Plk1) regulates DNA replication origin firing and interacts with Rif1 in Xenopus.

Ciardo D , Haccard O , Narassimprakash H , Cornu D , Guerrera IC , Goldar A , Marheineke K .

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The activation of eukaryotic DNA replication origins needs to be strictly controlled at multiple steps in order to faithfully duplicate the genome and to maintain its stability. How the checkpoint recovery and adaptation protein Polo-like kinase 1 (Plk1) regulates the firing of replication origins during non-challenged S phase remained an open question. Using DNA fiber analysis, we show that immunodepletion of Plk1 in the Xenopus in vitro system decreases replication fork density and initiation frequency. Numerical analyses suggest that Plk1 reduces the overall probability and synchrony of origin firing. We used quantitative chromatin proteomics and co-immunoprecipitations to demonstrate that Plk1 interacts with firing factors MTBP/Treslin/TopBP1 as well as with Rif1, a known regulator of replication timing. Phosphopeptide analysis by LC/MS/MS shows that the C-terminal domain of Rif1, which is necessary for its repressive action on origins through protein phosphatase 1 (PP1), can be phosphorylated in vitro by Plk1 on S2058 in its PP1 binding site. The phosphomimetic S2058D mutant interrupts the Rif1-PP1 interaction and modulates DNA replication. Collectively, our study provides molecular insights into how Plk1 regulates the spatio-temporal replication program and suggests that Plk1 controls origin activation at the level of large chromatin domains in vertebrates.

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Species referenced: Xenopus laevis
Genes referenced: cdc45 cdc7 chek1 ctrl lss mcm4 mtbp npy4r orc2 plk1 rif1 ticrr topbp1
GO keywords: DNA replication checkpoint [+]
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	Graphical Abstract
	Figure 1. Plk1 depletion decreases replication fork density and initiation frequency during unchallenged S phase. (A) Xenopus egg extracts were mock- or Plk1-depleted and remaining Plk1 was quantified on western blot using anti-Plk1 antibody. * marks a non-specific band. (B) Sperm nuclei (2000 nuclei/μl) were incubated in mock- or Plk1-depleted Xenopus egg extracts in the presence of Biotin-dUTP for different times and analyzed by DNA combing. Selection of three representative DNA fibers of early S phase from mock- (top three) or Plk1-depleted (bottom three) extract; in red, replication eyes labeled by Biotin-dUTP, in green, whole DNA fibers. (C) Replicated fraction (%), fork density (kb–1) and frequency of initiation (kb–1s–1) for three independent experiments at 2 or 3 time points were calculated and reported in the table. (D) scatter blot of ratios Δ Plk/Δmock of replication parameters for each line shown in C, with mean (n = 8) and standard deviation, P values, one sample t-test, compared to theoretical mean 1.
	Figure 2.Plk1 depletion increases eye-to-eye distances, independently of replicated fraction of DNA fibers. Sperm nuclei (2000 nuclei/μl) were incubated for different times in mock- (ΔM, dark grey) or Plk1-depleted (ΔP, blue) Xenopus egg extracts in the presence of Biotin-dUTP. (A) Eye-to-eye distance distribution (n = 77–1151) and (B) eye length distribution (n = 196–2507) (kb) (scatter dot plots with median) of one combing experiment representative of middle and late S phase. All DNA fibers were then grouped together in different bins according to their replicated fraction. Means (n = 51–1189) with SEM of (C) fork density (kb–1) and (D) eye-to-eye distances (kb), calculated for the different bins of replicated fractions of all fibers. * indicates significant difference (Mann–Whitney U test, two-sided, P< 0.05: P-values: * 0.01–0.05; 0.001–0.01; * 0.0001–0.001; ****<0.0001).
	Figure. 3. Numerical simulations: Plk1 depletion decreases the fraction of the genome with high probability origin firing, θ, and the rate of import of limiting factors, J. (A) Diagram of numerical model from (29): Potential replication origins located in a fraction θ of the genome have a probability of firing Pin higher than probability of firing Pout of potential origins located in the complementary genome fraction 1 - θ. The firing of a potential origin requires its encounter with limiting factors whose number N(t)=N0+Jt increases as S phase progresses. The origin firing probability over a distance d ahead of a replication fork is enhanced of a probability Plocal⁠. (B–H) Inferred model parameters by fitting Δmock and ΔPlk1 depleted combing data for two different time points as indicated with replicated fraction: circles indicate the mean value of the parameters over 100 different runs of the genetic algorithm and the error bar are standard deviations, solid lines are the mean (n = 2) values of consecutive parameters whose differences are not statistically different, (*) indicates significant difference between the Δmock and ΔPlk samples, student t-test as described in Appendix 2 of (29), (⁠X2>1, P values < 10–7).
	Figure 4. Dynamics of chromatin bound proteins after Plk1 depletion by LC/MS/MS. Sperm nuclei (4000 nuclei/μl) were allowed to replicate in Xenopus egg extracts, chromatin was isolated and analyzed according the Chromass protocol (Materials and Methods). Heat map of significant (P< 0.05) increased or decreased proteins in Plk1 depleted samples versus control samples (A, B, C are triplicates). Protein MS1 intensities are normalized across samples using z score: red indicates intensity above average, bright green below. For each protein, GO-term functions retrieved from Uniprot database manually curated. Plk1.L and Plk1.S signifies proteins encoded by the two different subgenomes L and S of X. laevis.
	Figure 5. Plk1 depletion stabilizes the binding of Treslin, MTBP and TopBP1 on chromatin. Sperm nuclei (2000 nuclei/μl) were added to mock- and Plk1-depleted egg extracts. Isolated chromatin was subjected to gel electrophoresis and western blot analysis using the indicated antibodies. (A) Representative western blot at 70 min for mock-depleted extracts, Plk1-depleted extracts and Plk1-depleted extracts replenished with recombinant Plk1 (300 nM). (B) Mean fold increase with SEM of MTBP, TopBP1, and Treslin optical density of bands in western blots (OD) normalized to Orc2 in six independent experiments at mid S phase (60–70 min) for depletions (n = 8), three experiments for add back with recombinant Plk1 (n = 3). * indicates significant differences, ns not significant, two-sided, unpaired t-test. (C) As in (A) but chromatin fractions were isolated at indicated times, in parallel with combing experiment from Figure 2.
	Figure 6. Plk1 co-immunoprecipitates with MTBP, Treslin and TopBP1. (A) Egg extracts were incubated with control (IP Ctrl) or Plk1 (IP Plk1) antibody coupled Sepharose beads. Immunoprecipitates and input extract were subjected to gel electrophoresis and western blot analysis using the indicated antibodies. (B) Chromatin fractions, 60 min after the addition of 2000 nuclei/μl in mock- or Plk1-depleted extracts, analyzed by western blot with indicated antibodies. (C) Chromatin fractions, isolated 60 min after the addition of 2000 nuclei/μl in egg extracts were treated or not with λ phosphatase and subjected to western blotting with indicated antibodies. (D) Sperm nuclei were incubated in egg extracts in the absence or presence of the ATR/ATM inhibitor caffeine (5 mM) and the Chk1 inhibitor AZD7762 (0.5 μM), reactions were stopped at indicated times, chromatin was purified and subjected to western blotting with indicated antibodies. (E) Sperm nuclei were incubated in egg extracts in the absence or presence of the Cdc7 inhibitor PHA767491 (50 μM), reactions were stopped at 60 min, chromatin was purified and subjected to western blotting with indicated antibodies.
	Figure 7. ChIP mass spectroscopy reveals potential Plk1 interactions with DNA replication proteins. (A) Experimental diagram of the ChIP MS approach from the Xenopus in vitro system after 15 min (pre-RC assembly) and 60 min (ongoing replication) incubation of sperm nuclei (4000 nuclei/μl) in egg extracts. (B) Identified nuclear proteins enriched more than 3-fold (spectral counts) in Plk1- versus mock-ChIP were classified into various functional groups according to GO-term location and functions retrieved from Uniprot database manually curated.
	Figure 8. Plk1 interacts with Rif1 and phosphorylates the C-terminal region of Rif1. (A) Left: Chromatin was purified after 70 min incubation of sperm nuclei (2000 nuclei/μl) in extracts depleted of mock, Plk1 or Plk1 replenished with recombinant Plk1 (300 nM) (ΔP + Plk1) and analyzed by western blotting, right: mean (n = 17 and 8) fold change with SEM of Orc2 normalized Rif1 signal compared to mock depletions from four different experiments, Wilcoxon signed rank test, * indicates significant and ns non-significant differences. (B) Cytoplasmic extract, LSS (Input) was incubated with control or Plk1 antibody coupled to Sepharose beads (respectively IP Ctrl and IP Plk1) or with control or Rif1 antibody coupled to Sepharose beads (respectively IP Ctrl and IP Rif1). The input and immunoprecipitates were subjected to gel electrophoresis and western blot analysis using the indicated antibodies. (C) In vitro phosphorylation of recombinant Rif1–CTD (6 μM) or control substrate casein (6 μM) by recombinant Plk1 (60 nM) in the presence of [γ32P]-ATP with or without the Plk1 inhibitor BI2536 (100 nM); left, representative autoradiography of SDS-PAGE; right, mean with SD of relative phosphorylation of three independent experiments, t-test, P value 0.005. (D) Phosphorylation of Rif1–CTD in S phase egg extracts: Rif1–CTD was added to mock- or Plk1- depleted extract in the presence of [γ 32P]-ATP, purified using Ni-Sepharose beads and analyzed on SDS-PAGE. Top, autoradiography; middle, Coomassie stain; bottom, mean (n = 2) with SEM of relative phosphorylation of Rif1–CTD normalized to total Rif1–CTD pulled down (Coomassie). (E) Identified phosphopeptides and modified amino acids in Rif1–CTD phosphorylated in vitro by recombinant Plk1, as in (D), but in the presence of cold ATP, by LC/MS/MS analysis. (F) Diagrammatic representation of Xenopus Rif1 with Rif1–CTD composed of three C-terminal regions (CR) comprising PP1 binding motifs SILK and RVSF located in CRI, adapted from (74).
	Figure 9. Rif1–CTD accelerates DNA replication dependent on PP1 interaction with Rif1-S2058. (A) Rif1–CTD or buffer was added to sperm nuclei (2000 nuclei/μl) replicating in egg extracts in the presence of [α 32P]-dCTP, DNA was purified at indicated times and replication was analyzed on alkaline gel electrophoresis, the mean incorporation with SEM from two independent experiments was plotted. (B) Rif1–CTD or buffer was added to sperm nuclei replicating in egg extracts, chromatin was purified at indicated times, specified proteins were analyzed by western blotting. (C) Buffer (-), wild type (WT), 1 μM Rif1–CTD mutants respectively (S2058D) and (S2058A) were added to egg extracts, recovered by pull-down after 1h incubation using Ni-magnetic beads, then analyzed by western blotting using anti-PP1 (PP1γ) or anti-Rif1 (Rif1–CTD) antibodies. (D) PP1 bound to Rif1–CTD was quantified, normalized to Rif1, and expressed as mean fraction with SEM of PP1 bound to WT Rif1–CTD in three independent experiments, unpaired or one sample t-test. (E) As in (A) WT, S2058D and S2058A Rif1–CTD were incubated in replicating reactions and [α 32P]-dCTP incorporation was quantified after DNA gel electrophoresis then plotted as mean fold increase compared to control (buffer) with SEM, in 3 (for WT, S2058D) or 5 (for WT, S2058A) independent experiments with different time points within (n = 9, n = 15 respectively), Mann-Whitney test. (F) Rif1–CTD or buffer was added to sperm nuclei (2000 nuclei/μl) replicating in mock- (Δmock), or Plk1- (ΔPlk1) depleted extracts, chromatin was purified after 60 min and indicated proteins were analyzed by western blotting. (G) Fold increase of chromatin bound PP1 normalized to Orc2 OD on western blots in Plk1- versus mock- depleted extracts from five different replication reactions with different time points, scatter dot blot with mean (n = 13), Wilcoxon signed ranked test, P-value above blot. (H) Model of Plk1 regulating origin activation via phosphorylation of Rif1 which interrupts the Rif1-PP1 interaction.
	Supplementary Figure S1 Plk1 depletion induces a decrease in replication extent, fork density and initiation frequency in three independent DNA combing replicates. Plots from values presented in Figure 1C, analysis performed as described in Material and Methods Section.
	Supplementary Figure S3 Differences in EL distribution and of mean frequency of initiation and EL dependent on replicated fraction of DNA fibers. (A) Eye length distributions, in experiment of Fig. 2 A and B, in mock and Plk1 depleted conditions, the distributions are different and shifted to bigger eyes after Plk1 depletion. (B) Single fibers were grouped in different bins in accordance to their replicated fraction. The figures represent the mean with SEM of frequency of initiation (B) and eye length (EL) (C) calculated for the different bins of replicated fraction, * indicates significant difference between mock and Plk1 depleted condition (Mann-Whitney U test, p<0.05).
	Supplementary Figure S4 Supplementary combing analysis. (A) fraction of fibers with only 0 % or 100 % replication per fiber in mock and Plk1 depleted conditions of experiment in Fig. 2A, B. (B) distribution of replicated fraction per fibers in mock and Plk1 depleted conditions (except fibers with 0 and 1 replicated fractions) in three time points. The graphs show over-representation of low and high replication extents after Plk1 depletion. (C) 0.2 Distributions of number of eyes pe0r.2fiber. It shows that after Plk1 depletion fibers with more 0th.1an 5-7 eyes are underrepresented , P values from Mann-Whitney U-test.
	Modeling experimental combing data with MM4 model (Ciardo et al. 2020a) in the case where the potential origins are heterogeneously distributed along the genome. Open circles are experimental data for different replication parameters; the red dashed line is the average of 100 independent fits from MM4 and the red error bars indicate standard deviations. [see suppl. files for equations]
	Supplementary Figure S6 Plk1 depletion decreases Cdc45 and increases TMT levels on chromatin. Quantification of optical densities (OD) of blots in Fig.5 C, normalized to Orc2 levels.
	Supplementary Figure S7 Effect of ATR/ATM and Chk1 inhibition separately on chromatin bound MTBP. Same experiment than in Fig. 6D but inhibitors (caffeine (ATR/ATMi) and AZD7762 (Chk1i)) were added in separated replication reactions and chromatin bound proteins were isolated and revealed in western blotting with indicated antibodies
	Supplementary Figure S8 DDK inhibition leads to a mobility shift of chromatin bound MTBP and Treslin throughout S phase. Complete time course experiment of Fig.6E, showing additional time points, 10, 30 and 90 min to better appreciate the reproducibility of mobility shifts during S phase.
	Supplementary Figure S9 Plk1 interacts with Rif1. Nucleoplasmic extract (Input) was incubated with (A) control (IP Ctrl) or Plk1 antibody coupled to Sepharose beads (IP Plk1) or with (B) control (IP Ctrl) or Rif1 antibody coupled to Sepharose beads (IP Rif1). The input and immunoprecipitates were subjected to gel electrophoresis and western blot analysis using the indicated antibodies.
	Supplementary Figure S10 Plk1 phosphorylates Rif1 in vitro. Stain-Free detection (Bio-Rad) as loading control of the phosphorylation reaction shown on Fig. 8 C. Samples were withdrawn from the assembled reaction just prior to [g32P]-ATP addition and run on SDS-PAGE.
	Supplementary Figure S11 Effect of Rif1-CTD WT (wild-type), S2058D and S2058A mutant on chromatin-bound PP1 and MCM4. Experiment as in Fig.9C, but equivalent amounts of WT or mutants were added to replication reactions, chromatin was purified after 60 min and immuno-blotted with indicated antibodies, Histone H3 as loading control.

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