Errico A, Cosentino C, Rivera T, Losada A, Schwob E, Hunt T, Costanzo V.

Cancer Research UK, 206281 European Research Council, ERC_206281 European Research Council

	Figure 1. Tipin is required for efficient DNA replication under ‘minimum licensing' conditions. (A) Immunoblot to assess Tipin depletion from egg extract. (B) Time course of DNA replication in ‘minimum licensed', mock- (blue bar) and Tipin-depleted extract (red bar) (right panel). (C) The efficiency of DNA replication in ‘minimum licensed', mock- (lane 1) and Tipin-depleted extract (lane 2) at 120 min post nuclei addition was tested. The defect in DNA replication observed in the Tipin-depleted extract (lane 2) was rescued by the addition of Tipin/Tim1 recombinant protein complex (lane 3) or mock extract (lane 4). Three independent experiments are averaged in the bar graphs. The error bars are standard deviation from the mean value.
	Figure 2. Tipin is required for Polα loading on the chromatin in ‘minimum licensing' condition. (A) Xenopus extract was immunoprecipitated with either anti-Tipin or preimmune serum. Samples were probed with anti-Polα and anti-Tipin antibodies. (B) Immunoblot analysis to detect the level of Polα, Orc1 and Tipin in total extract (mock or Tipin depleted). (C) Immunoblot to detect the level of Polα, Orc1, cdc45, Sld5 and Tipin on the chromatin, at different time points (30, 60, 90 min), under ‘maximum' (right panel) or ‘minimum' licensing (left panel) condition.
	Figure 3. Analysis of inter-origin distance in the Tipin-depleted extract by molecular combing. (A) Scheme of the molecular combing experiment and visualization by immunofluorescence of DNA fibres. Green tracts represent origins of DNA replication (replication eye). (B) Distribution of inter-origins distance under ‘maximum' and ‘minimum' licensing condition in samples where biotin–dUTP was added at t=40 min or at t=60 min. The mock extract is represented by the red bar and the Tipin-depleted extract by the blue bar. Black arrows indicate a shift of the peak of distribution of inter-origins distances towards higher value in the ‘minimum licensed', Tipin-depleted extract at t=60 min. The difference between the distribution of inter-origin distance in the mock- and Tipin-depleted extract observed at 60 min in minimum licensing condition is statistically significant (t-test P<0.0001).
	Figure 4. Tipin, And1 and Polα directly interact. (A) Equal amounts of extract were immunoprecipitated with either anti-Tipin, anti-And1 antibodies or pre-immune serum. Purified proteins were immunoblotted with the indicated antibodies to detect associated proteins. (B) Pull-down assay using GST or GST–And1 glutathione–Sepharose beads and recombinant Tipin–6His. The presence of Tipin–6His was detected both with anti-Tipin or anti-6His antibody. (C) Pull-down assay using GST, GST–And1 or GST–Tipin and in vitro translated 35S-labelled Polα p180. Polα was detected by autoradiograph.
	Figure 5. Tipin and And1 are both required for DNA replication and for the efficient association of Polα to the chromatin. (A) The efficiency of DNA replication was measured in mock and depleted extracts (ΔTip, ΔAnd1, ΔAnd1/Tip). Three independent experiments are averaged in the bar graphs. The error bars are standard deviation from the mean value. (B) Sperm nuclei were added to Xenopus extract, mock or depleted (ΔTip, ΔAnd1), and chromatin was harvested at different times. Chromatin-bound proteins were analysed by SDS–PAGE and immunoblot analysis with the indicated antibodies. (C) Immunoblot analysis to detect the level of Polα, Orc1 and Mcm10 on the chromatin, at different time points, in mock and depleted extracts (ΔTip, ΔAnd1, ΔAnd1/Tip). Orc1 staining was used for normalization. (D) Immunoblot analysis to detect the level of Mcm7, TopBP1, Cdc45 on the chromatin, at different time points, in mock and depleted extracts (ΔTip, ΔAnd1, ΔAnd1/Tip). Histone H1-b4 staining was used for normalization.
	Figure 6. Tipin and And1 contribute to sister chromatid cohesion in S-phase. (A) Immunofluorescence analysis of mitotic chromosomes assembled in the indicated extract. Sister chromatids are stained with anti-condensin antibody, xCAPE (green). Biotin–dUTP was also added to the extracts to confirm that chromosome had replicated and detected with fluorescently labelled streptavidin (red). An enlarged section of the chromatids (boxed area) is also represented. (B) Distribution of the distance between sister chromatids in the different extracts.
	Figure 7. Model for Tipin–And1–Polα function. Tipin binds directly to And1 and Polα. And1 also binds Polα and GINS directly. Tipin/Tim1/And1 might create a flexible bridge between replisome components such as Cdc45, GINS, Polα and the MCM complex necessary for the stable binding of Polα to the replication fork. The cohesin ring is also represented.

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