Kumagai A , Shevchenko A , Shevchenko A , Dunphy WG .

GM043974 NIGMS NIH HHS , GM070891 NIGMS NIH HHS , R01 GM070891-05 NIGMS NIH HHS , R37 GM043974-18 NIGMS NIH HHS , R01 GM070891 NIGMS NIH HHS , R37 GM043974 NIGMS NIH HHS , R01 GM043974 NIGMS NIH HHS

	Figure 1. Identification of Treslin as a TopBP1-Interacting Protein in Xenopus Egg Extracts (A) Nuclear lysates were incubated without (lane 1) or with HF-TopBP1(I-VI) (lane 2) in the presence of anti-FLAG antibody beads. Beads were retrieved and processed for silver staining. (B) Interphase egg extract (lane 1) and recombinant Xenopus Treslin from insect cells (lane 2) were immunoblotted with anti-Xenopus Treslin antibodies. Lanes are aligned from the same gel. (C) Nuclear lysates from egg extracts lacking (lanes 2, 4, and 6) or containing aphidicolin (APH) (lanes 3, 5, and 7) were incubated with anti-FLAG beads containing no recombinant protein (lanes 4 and 5) or full-length HF-TopBP1 (lanes 6 and 7). Beads were reisolated and immunoblotted for Treslin and TopBP1. Lane 1 shows an aliquot of initial egg extract. (D) M phase extracts (M) from unactivated eggs were left unactivated (lanes 1, 8, and 15) or activated with calcium (lanes 2–7, 9–14, 16, and 17). Extracts were incubated with (lanes 1–14) or without sperm nuclei (lanes 15–17). Chromatin fractions from activated extracts were isolated at the indicated times. Chromatin from M phase extracts was incubated for 30 min (lanes 8 and 15). Mock chromatin fractions were isolated from extracts lacking sperm nuclei (lanes 15–17) at the indicated times. Samples were immunoblotted for the indicated proteins. (E) Egg extracts were incubated in the presence (lanes 1–4 and 6–9) or absence of sperm nuclei (lane 5) with no inhibitor (lanes 1, 5, and 6), geminin (lanes 2 and 7), p27 (lanes 3 and 8), or both geminin and p27 (lanes 4 and 9). Soluble nuclear fractions (lanes 1–4) and chromatin fractions (lanes 5–9) were immunoblotted for the indicated proteins. See also Table S1.
	Figure 2. Treslin Interacts with a Region of TopBP1 that Is Essential for DNA Replication (A) Regions of TopBP1 necessary for DNA replication. Egg extracts (lane 1) were mock depleted with control antibodies (lane 2) or immunodepleted with anti-TopBP1 antibodies (lanes 3–10). TopBP1-depleted extracts were supplemented with no recombinant protein (lane 3), full-length (WT) HF-TopBP1 (lane 4), or deletion mutants of TopBP1 lacking the indicated BRCT domains or the ATR-activating domain (AAD) (lanes 5–10). DNA replication was determined by incorporation of 32P into chromosomal DNA after 120 min. Labeling of both the upper and lower bands was quantitated. (B) Egg extracts (lane 1) were subjected to mock depletion (lane 2) or immunodepletion of TopBP1 (lanes 3–8). TopBP1-depleted extracts were supplemented with no recombinant protein (lane 3), full-length HF-TopBP1 (lane 4), or fragments containing BRCT domains I-II (lane 5), III (lane 6), or I-III (lane 8). For lane 7, two fragments containing domains I-II or III were added together. (C) Quantitation of the data from (A) and (B). Results were compiled from seven independent experiments. Values are from at least two independent experiments (mean ± standard deviation). Replication in mock-depleted extracts is denoted as 100%. (D) Nuclear lysates from egg extracts containing no tagged protein (lane 1) or the indicated versions of HF-TopBP1 (lanes 2–6) were immunoprecipitated with anti-FLAG beads. Samples were processed for immunoblotting with anti-Treslin (top) and anti-FLAG antibodies (bottom). (E) Nuclear lysates (lane 1) from egg extracts containing no tagged protein (lane 2) or the indicated fragments of TopBP1 (lanes 3–5) were incubated with anti-FLAG beads. Beads were retrieved and immunoblotted with anti-Treslin (top) and anti-FLAG antibodies (bottom). Asterisk denotes cross-reacting IgG heavy chain. See also Figure S1.
	Figure 3. Treslin Is Required for Chromosomal DNA Replication in Xenopus Egg Extracts (A) Egg extracts (lane 1) were mock depleted with control antibodies (lane 2) or immunodepleted with anti-Treslin antibodies (lane 3) and immunoblotted for Treslin. (B) Mock-depleted (lanes 1–6) and Treslin-depleted (lanes 7–12) extracts were assayed for chromosomal DNA replication at the indicated times. (C) Compilation of results (mean ± standard deviation) from four independent experiments on the extent of replication in mock-depleted (left) and Treslin-depleted (right) extracts at the indicated times. Replication at 120 min in mock-depleted extracts is denoted as 100%. (D) Mock-depleted extracts (lanes 1, 5, and 6), Treslin-depleted extracts (lanes 2 and 7), TopBP1-depleted extracts (lanes 3 and 8), and TopBP1-depleted extracts supplemented with HF-TopBP1(I-III) (lanes 4 and 9) were incubated for 90 min in the absence (lane 5) or presence (lanes 6–9) of sperm nuclei. Extracts (lanes 1–4) and chromatin fractions (lanes 5–9) were immunoblotted for the indicated proteins. (E) Chromatin fractions from mock-depleted (lane 1), Treslin-depleted (lane 2), and TopBP1-depleted (lane 3) extracts were immunoblotted for the indicated proteins. See also Figure S2.
	Figure 4. Association of Treslin with TopBP1 Depends Upon Cdk2 (A) Nuclear lysates were prepared from egg extracts incubated without (lanes 1, 3, 4, 6, and 7) or with p27 (lanes 2, 5, and 8). Input lysates (lanes 1 and 2), control immunoprecipitates (lanes 3 and 6), and anti-TopBP1 immunoprecipitates (lanes 4, 5, 7, and 8) were immunoblotted for Treslin and TopBP1. Immunoprecipitations were carried out in the absence (lanes 3–5) or presence of 50 μg/ml ethidium bromide (EtBr) (lanes 6–8). (B) Nuclear lysates were prepared from egg extracts incubated without (lanes 1, 3, and 4) or with p27 (lanes 2 and 5). Input lysates (lanes 1 and 2), control immunoprecipitates (lanes 3), and anti-Treslin immunoprecipitates (lanes 4 and 5) were immunoblotted for Treslin and TopBP1. (C) Nuclear lysates from egg extracts lacking (lanes 1, 3, 4, and 6) or containing p27 (lanes 2, 5, and 7) were incubated with anti-FLAG antibody beads containing no tagged protein (lane 3) or the indicated fragments of TopBP1 (lanes 4–7). Beads were retrieved and immunoblotted with anti-Treslin and anti-FLAG antibodies. Lanes 1 and 2 depict input nuclear lysates. (D) Nuclear fractions were prepared from extracts that were mock treated (lane 1) or treated with geminin (lane 2) or p27 (lane 3). Samples were immunoblotted for Treslin. (E) Nuclear lysates (lane 1) containing no tagged protein (lane 2) or the HF-TopBP1(I-III) fragment (lanes 3–5) were incubated with anti-FLAG beads. Beads were retrieved and incubated without (lanes 2 and 3) or with 20 U/μl lambda phosphatase (lanes 4 and 5) in the absence (lane 4) or presence of 10 mM sodium vanadate (lane 5) for 30 min at room temperature. Vanadate is an inhibitor of lambda phosphatase. Samples were immunoblotted for Treslin. (F) Eluates of chromatin treated with 0.45 M NaCl (lane 1) were incubated in the absence (lanes 2 and 6) or presence of lambda phosphatase (lanes 3 and 7) for 30 min at room temperature or left on ice (lanes 4 and 5). Subsequently, eluates were mixed with anti-FLAG beads in the absence (lane 4) or presence of HF-TopBP1(I-III) (lanes 5–7). Beads were retrieved and immunoblotted with anti-Treslin (top) or anti-FLAG antibodies (bottom). See also Figure S3.
	Figure 5. Depletion of Treslin from Human Cells Results in Defective S Phase (A) Human U2OS cells were treated with control siRNA (lanes 1–3) or Treslin siRNA #1 (lanes 4–6) for 48, 72, and 96 hr. Cell lysates were immunoblotted for Treslin, α-tubulin, and γ-H2AX. (B) Cell numbers in control and Treslin siRNA-treated cultures were counted at indicated times after transfection. (C) DNA content of cells treated with siRNA was analyzed by flow cytometry. Cells were stained with propidium iodide. DNA content is shown on the x axis. The y axis indicates the relative number of cells. (D) Cells treated with control or Treslin siRNA were incubated in the absence or presence of BrdU for 60 min at 72 hr after transfection. Intensity of BrdU incorporation is indicated on the logarithmic y axis; DNA content is shown on the x axis. The boxed area indicates cells incorporating BrdU. See also Figure S4.
	Figure 6. Treslin-Depleted Cells Defective for DNA Replication Also Accumulate Damaged DNA (A) Cells treated with control siRNA or Treslin siRNA #1 for 72 hr were stained with Hoechst dye to detect DNA, Click-iT reagent to detect incorporated EdU, and antibodies against γ-H2AX. (B) Chromatin fractions were prepared at 48 and 72 hr from U2OS cells transfected with control siRNA (lanes 1 and 2) or Treslin siRNA #1 (lanes 3 and 4). Fractions were immunoblotted for Cdc45 (top) and Orc2 (bottom). (C) Control (lanes 1 and 2) and Treslin siRNA-treated cultures (lanes 3 and 4) were incubated for 15 min in the absence (lanes 1 and 3) or presence of 10 μg/ml aphidicolin (lanes 2 and 4). Cultures were processed for immunoblotting with antibodies that detect Chk1 or phosphorylation of Chk1 on Ser345 or Ser317. See also Figure S5.
	Figure 7. Model for the Role of Treslin in DNA Replication See main text for details.
	Highlights: Treslin is a newly identified factor for initiation of vertebrate DNA replication ► Treslin interacts with TopBP1, a protein required for initiation of DNA replication ► Association of Treslin and TopBP1 requires cyclin-dependent kinase activity ► Treslin and TopBP1 collaborate in loading the initiator protein Cdc45 onto DNA
	Figure S2. Immunodepletion of Treslin Does Not Irreversibly Compromise the Functional Integrity of Egg Extracts—Related to Figure 3 (A) Nuclei in mock-depleted and Treslin-depleted extracts were visualized at 90 min by phase-contrast microscopy (top). Scale bar indicates 10 μm. Nuclear diameter (mean ± standard deviation) was determined using the ImageJ program (bottom). More than 100 nuclei were measured in each sample in two independent experiments. (B) Mock-depleted (lane 1), Treslin-depleted (lane 2), and Treslin-depleted extracts that had been supplemented with salt extracts of replicating nuclei (lane 3) were immunoblotted for Treslin. (C) The extracts from panel B were assayed for chromosomal DNA replication at indicated times. As expected, the salt extract from replicating nuclei was incapable of DNA replication by itself (data not shown).
	Figure S3. Time-Course for Binding of Treslin and TopBP1—Related to Figure 4 M-phase extracts (lanes 1 and 7) and interphase extracts containing sperm nuclei (lanes 2-6 and 8-11) were incubated for the indicated times after activation with calcium. Extracts were diluted with buffer to a final concentration of 0.5% NP-40 and 0.4 M NaCl to dissolve nuclear membranes and release Treslin and TopBP1 from chromatin. Samples were then diluted and immunoprecipitated with control (lane 6) or anti-TopBP1 antibodies (lanes 7-11). Extracts and immunoprecipitates were immunoblotted for Treslin and TopBP1.
	Figure S4. Multiple Treslin siRNAs Elicit a Similar Inhibition of DNA Replication in Human Cells—Related to Figure 5 (A) Lysates from U2OS cells treated with control siRNA and the indicated Treslin siRNAs for 72 hr were immunoblotted for Treslin. Asterisks denote cross-reacting bands. (B) U2OS cells treated with the indicated siRNAs for 72 hr were labeled with EdU for 1 hr and stained with the Click-iT EdU flow cytometry staining kit. DNA was stained with 7-AAD. The y axis indicates fluorescence intensity, and the x axis indicates DNA content. (C) U2OS cells were treated with the indicated siRNAs for 72 hr and labeled with EdU for 1 hr. Cells were fixed and processed for detection of DNA (top), EdU (middle), or γ-H2AX (bottom).
	Figure S5. Rescue of DNA Replication in Treslin-Ablated Cells with Recombinant Treslin—Related to Figure 6 (A) U2OS T-REx cells capable of expressing an siRNA-resistant form of Treslin in a doxycycline-inducible manner were incubated for 24 hr in the absence (lanes 1 and 3) or presence of doxycycline (DOX) (lanes 2 and 4). The cells were then transfected with control siRNA (lanes 1 and 2) or Treslin siRNA #1 (lanes 3 and 4). After an additional 72 hr, cell lysates were prepared and immunoblotted for Treslin. (B) Quantitation of the percentage of cells incorporating EdU under the indicated conditions. Values were obtained from three independent experiments (mean ± standard deviation). (C) The indicated cultures were processed for visualization of incorporated EdU (top) or total DNA (bottom).

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