Trenz K , Errico A , Costanzo V .

Cancer Research UK

	Figure 1. Effects of Plx1 depletion on DNA replication. (A) Immunodepletion of Plx1. Western blot analysis with anti-Plx1 antibodies of control (C) mock-depleted (M), Plx1-depleted (ΔPlx1) and Plx1-depleted extracts reconstituted with 6 ng/μl recombinant Plx1 (+Plx1). (B) Replication assay in unchallenged extracts. Extracts supplemented with 2000 nuclei/μl were untreated (CTRL), mock depleted (Mock), Plx1 depleted (ΔPlx1) and Plx1 depleted and supplemented with recombinant Plx1 (+Plx1). DNA was replicated in the presence of [α-32P]dATP for 120 min, extracted and separated on an agarose gel. The gel shown represents a typical result. (C) Chromatin binding of Mcm7. Sperm nuclei were replicated in untreated extracts (CTRL) and extracts supplemented with a low concentration (40 nM) of geminin (GEM). Chromatin was isolated at the indicated time points and separated on SDS–PAGE. Western blots were performed with antibodies against human Mcm7. (D) Effects of geminin on DNA replication. Extracts were supplemented with increasing concentrations of geminin (40, 60 and 120 nM). (E) Phosphorylation of Chk1 serine 345 (p-CHK1) in extracts supplemented with 7000 nuclei/μl. Extracts were untreated (−) or supplemented (+) with 40 nM geminin (GEM). Western blot was performed with anti-phospho-serine 345 of human Chk1 (p-Chk1) and anti-Chk1. Chk1 phosphorylation induced by geminin was detected with ECL plus reagent (see Materials and methods). (F) Replication assay in extracts supplemented with 40 nM geminin (GEM) in the presence of 7000 nuclei/μl. Extracts were untreated (CTRL), mock depleted (Mock), Plx1 depleted (ΔPlx1) and Plx1 depleted and supplemented with recombinant Plx1 (Plx1-dep+Plx1). The gel shown represents a typical result.
	Figure 2. Plx1 binding to chromatin is enhanced in the presence of stalled replication forks. (A) Chromatin binding of Plx1 and Mcm7 to chromatin 30, 60 and 90 min after nuclei addition to untreated extracts (CTRL) and extracts treated with geminin (GEM) or 5 μM aphidicolin (APH). (B) Chromatin binding of Plx1 after aphidicolin treatment. Extracts were untreated (CTRL) or treated with 5 μM aphidicolin (APH) before the addition of sperm nuclei. Chromatin-bound proteins were separated on SDS–PAGE, transferred to nitrocellulose and probed for Mcm7 and Plx1, which were visualized with infrared dye-labelled secondary antibodies. (C) Quantification of chromatin-bound Plx1 at 30 and 60 min after nuclei addition to untreated (CTRL) or aphidicolin (APH)-treated extracts. Chromatin-bound proteins were quantified with an infrared imager and the ratio of Plx1 to Mcm7 was plotted. The ratio Plx1 to Mcm7 in the untreated sample (CTRL) was considered as the reference value.
	Figure 3. Plx1 suppresses the caffeine-sensitive intra-S-phase checkpoint. (A) The effects of caffeine on DNA replication under stress in the absence of Plx1. Sperm nuclei (7000 nuclei/μl) were replicated in extracts that were untreated (CTRL), mock depleted (Mock) or Plx1 depleted (ΔPlx1) in the presence of 2.5 μM aphidicolin (APH) or 40 nM geminin (GEM). Extracts were supplemented with buffer (Buff) or 5 mM caffeine (Caff). (B) Phosphorylation of Chk1 in untreated (CTRL), mock-depleted (Mock) and Plx1-depleted (ΔPlx1) extracts that were untreated (−) or supplemented with 50 ng/μl pApT (+). Western blot was performed with anti-phospho-Chk1 (p-Chk1), anti-Chk1 and anti-Xorc1 antibodies. Quantification was made by measuring optical density (OD) and is presented as a bar graph. (C) Chk1 phosphorylation in nuclei. Sperm nuclei were incubated with untreated extracts (CTRL) or with Plx1-depleted extracts (ΔPlx1). All extracts were untreated (−) or supplemented with 50 μM aphidicolin (+). The nuclei were isolated and the nuclear proteins were separated by SDS–PAGE. Western blot was performed with anti-phospho-Chk1 (p-Chk1), anti-Chk1 and anti-Xorc1 antibodies. (D) Sperm nuclei were incubated with untreated extracts (CTRL) or with extracts supplemented with 6 ng/μl Plx1 (Plx1) and processed as described above. All extracts were untreated (−) or supplemented with 50 μM aphidicolin (+). As described above, quantification of western blot signals in panels C and D was made by measuring OD and is presented as bar graphs.
	Figure 4. Plx1 deficiency affects Cdc45 loading and Cdk2 activity in the presence of stalled replication forks. (A) Cdc45 and Mcm7 binding to chromatin isolated from extracts that were untreated (CTRL), Plx1 depleted (ΔPlx1) or Plx1 depleted and reconstituted with recombinant Plx1 (+Plx1). All extracts were supplemented with aphidicolin. (B) Nuclear Cdk2 activity measured by monitoring 32P incorporation in histone H1 after immunoprecipitating nuclear Cdk2 with anti-Xenopus Cdk2 antibodies from extracts that were untreated (CTRL), mock depleted (Mock) or Plx1 depleted (ΔPlx1) and treated with and without 50 μM aphidicolin (APH) or 5 mM caffeine (Caff), as indicated.
	Figure 5. Plx1 interacts with the Mcm complex through its Polo box domain. (A) Immunodepletion of Tipin. Extracts were mock depleted (Mock) or Tipin depleted using anti-Tipin antibodies (ΔTipin). The cytosol was then subjected to western blot with anti-Tipin antibodies. (B) Plx1 and Claspin binding in Tipin-depleted extracts. Sperm nuclei were incubated in mock-depleted (Mock) or Tipin-depleted (ΔTipin) extracts in the presence of 2.5 μM aphidicolin (APH). Chromatin was isolated at the indicated time points and probed with anti-Plx1 and anti-Claspin antibodies. (C) Co-immunoprecipitation of Plx1 and Mcm7. Extracts that were untreated (−) or treated with 50 ng/μl pA/pT (+) were immunoprecipitated with pre-immune (Pre-Imm) or anti-Plx1 (Anti-Plx1) antibodies. Western blot was performed using anti-Mcm7 and anti-Plx1 antibodies. (D) Pull-down with GST–Polo box and MALDI-TOF analysis. Interphase extracts were incubated with GST–agarose beads or agarose beads with GST fused to the Polo box derived from human Plk1. After pull-down, proteins were consecutively eluted using 3 mg/ml of phosphopeptide solution (first elution=E1, second elution=E2, see Supplementary data for the sequence of the peptide and the Polo box) and subjected to MALDI-TOF analysis. Band 1 corresponds to Mcm2, band 2 to Mcm6, band 3 to Mcm4 and band 4 to Mcm7. The GST fusion protein containing the Polo box from Plk1 is shown at the bottom. (E) GST–Polo box pull-down of nuclear proteins probed with anti-Mcm7 antibodies (GST–Polo box). The extract used for the pull-down was supplemented with nuclei and buffer (Buff), 2.5 μM aphidicolin (APH) or 2.5 μM aphidicolin and 5 mM caffeine (Caff), as indicated. The total amount of unbound Mcm7 extracted from nuclei was loaded as a control (Input). (F) Effects of Mcm complex depletion on Plx1 binding to chromatin. The extract was untreated (C), mock depleted (M) or Mcm depleted (ΔMCM) using anti-Mcm3 antibodies and incubated with sperm nuclei for 60 min in the presence of 2.5 μM APH. Cytosol and chromatin were then subjected to western blot with anti-Mcm7 and anti-Plx1 antibodies.
	Figure 6. Mcm2 serine 92 phosphorylation is required for Plx1 function. (A) Chromatin binding of Plx1 in the presence of Mcm2-WT and Mcm2-S92A proteins. Sperm nuclei were incubated for 60 min in extracts supplemented with 200 ng/μl recombinant histidine-tagged Mcm2-WT and Mcm2-S92A proteins in the presence of 5 μM etoposide. Chromatin was isolated and subjected to western blot analysis with anti-Plx1 (Plx1) and anti-histidine (His-Mcm2) antibodies. (B) DNA replication under stress in Plx1-depleted extracts in the presence of Mcm2-WT and Mcm2-S92A proteins. Sperm nuclei (7000 nuclei/μl) were replicated in extracts that were untreated (CTRL), mock depleted (Mock) or Plx1 depleted (ΔPlx1) in the presence of 5 μM etoposide. Extracts were supplemented with buffer (Buff), 200 ng/μl Mcm2-WT or 200 ng/μl Mcm-S92A proteins. Plx1-depleted extracts were also supplemented with 6 ng/μl recombinant Plx1, as indicated.
	Figure 7. Plx1 prevents chromosomal breakage during DNA replication. (A) DNA breaks detected with the TUNEL assay. Egg extract was left untreated (CTRL), mock depleted (Mock), Plx1 depleted (ΔPlx1) or Plx1 depleted and supplemented with recombinant Plx1 (ΔPlx1 dep+Plx1). DNA replication was obtained in the absence (grey bar) or presence of 40 nM geminin (black bar). Sperm nuclei were replicated for 90 min, chromatin was isolated and TUNEL assay was performed in the presence of [α-32P]dATP. The DNA was isolated, precipitated and separated on an agarose gel. Incorporated 32P was quantified with the PhosphoImager and optical density (OD) was plotted. (B) Typical image of a nucleus processed with the neutral comet assay from mock-depleted extract (Mock) and from Plx1-depleted extract (ΔPlx1). (C) Quantification of DSBs. Untreated extracts (grey) and extracts supplemented with geminin (black) were left untreated (CTRL), mock depleted (Mock), Plx1 depleted (ΔPlx1) or Plx1 depleted and supplemented with recombinant Plx1 (ΔPlx1+Plx1). The percentage increase in the head length of the comets subtracted from the average nuclear diameter of undepleted samples with high Mcm was used as a parameter to measure DSBs. (D) Model representing the function of Plx1 in DNA replication under stressful conditions. In the presence of stalled replication forks, the ATR/ATM-dependent checkpoint activation promotes Plx1 binding to the Mcm complex close to stalled forks and attenuates the inhibitory activity of the checkpoint towards unfired origins. Activation of the supplementary origins overcomes the stalled replication forks. Claspin-dependent recruitment of Plx1 suppresses the checkpoint that controls mitosis onset in the presence of unreplicated DNA.

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