Lyu K , Kumagai A , Dunphy WG .

R01 GM043974 NIGMS NIH HHS , R01 GM070891 NIGMS NIH HHS , R37 GM043974 NIGMS NIH HHS

	Figure 1. Characterization of ETAA1 in Xenopus egg extracts. (a) Interphase egg extracts were immunoblotted with antibodies against Xenopus ETAA1 (lane 1). Recombinant, baculovirus-expressed HFS-ETAA1 protein (lane 2) was electrophoresed on the same gel and immunoblotted with anti-ETAA1 antibodies. (b) Concentration of ETAA1 in egg extracts. The indicated amounts (ng) of antigen used for the production of anti-ETAA1 antibodies (His10-tagged version of residues 374–820) were mixed with egg extracts. Mixtures were subjected to gel electrophoresis and immunoblotted with anti-ETAA1 antibodies. Amounts of egg extract correspond to: 1 μl (lanes 1–4); 0.5 μl (lane 5); and 0.2 μl (lane 6). (c) Interphase egg extracts were incubated without (lane 1) or with sperm chromatin (lanes 2–9) in the absence (lanes 1–5) or presence of APH (lanes 6–9). Chromatin fractions were isolated from the extracts at the indicated times and immunoblotted for ETAA1, RPA70, and Orc2 (loading control). Lane 10 depicts the initial egg extract.
	Figure 2. Effect of depletion of ETAA1 on APH-induced phosphorylation of Chk1 and RPA in egg extracts. (a) Egg extracts (lane 1) were mock depleted with control antibodies (lane 2) or immunodepleted with anti-Xenopus ETAA1 antibodies (lane 3) and immunoblotted for ETAA1 and TopBP1, as indicated. (b) Untreated (lanes 1–2), mock-depleted (lanes 3–4), and ETAA1-depleted egg extracts (lanes 5–6) were incubated for 90 min in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 150 μM APH. Nuclear fractions from the extracts were prepared and immunoblotted with anti-phospho-Chk1 (p-Chk1), anti-Chk1, anti-phospho-RPA32 (p-RPA32), and anti-RPA32 antibodies. Lane 7 depicts the initial egg extract. (c) Mock-depleted (lanes 1–4) and ETAA1-depleted egg extracts (lanes 5–8) were incubated for 90 min or 180 min in the absence (lanes 1, 2, 5, and 6) or presence (lanes 3, 4, 7, and 8) of 150 μM APH. Nuclear fractions were prepared and immunoblotted with anti-ETAA1 antibodies. Note that the space between lanes 5 and 6 is a blank lane. Lane 9 depicts the initial egg extract. (d) Untreated (lanes 1–3), mock-depleted (lanes 4–6), and ETAA1-depleted egg extracts (lanes 7–9) were incubated for 90 min in the absence of APH (lanes 1, 4, and 7) or the presence of either 15 μM (lanes 2, 5, and 8) or 150 μM APH (lanes 3, 6, and 9). Nuclear fractions from the extracts were prepared and immunoblotted with the indicated antibodies. Lane 10 depicts the initial egg extract.
	Figure 3. Role of ETAA1 in DNA replication and related processes. (a) Mock-depleted (lanes 1–3) and ETAA1-depleted egg extracts (lanes 4–6) containing APH were incubated in the absence of sperm chromatin for 45 min (lanes 1 and 4) or the presence of sperm chromatin (lanes 2–3 and 5–6) for the indicated times. Incubations were processed for the preparation of chromatin fractions and the resulting samples were immunoblotted for Mcm2, Cdc45, and Orc2. Lane 7 depicts an aliquot of the initial egg extract. (b) Mock-depleted and ETAA1-depleted egg extracts were assayed for chromosomal DNA replication with 32P-labeled dATP at the indicated times as indicated. An agarose gel containing radiolabeled replication products (top) and corresponding quantitation (bottom) are depicted. Values are normalized to the amount of DNA replication at 90 min in mock-depleted extracts. Representative of three experiments. (c) Mock-depleted (lanes 1–5) and ETAA1-depleted egg extracts (lanes 6–10) were incubated in the absence (lanes 1 and 6) or presence of sperm chromatin (lanes 2–5 and 7–10). In addition, extracts either lacked (lanes 2, 4, 7, and 9) or contained APH (lanes 1, 3, 5, 6, 8, and 10). After incubation for the indicated times, extracts were processed for the preparation of chromatin fractions and the resulting samples were immunoblotted for BLM, TopBP1, Mre11, RPA70, and Orc2. Lane 11 depicts an aliquot of the initial egg extract. Note that the space between lanes 7 and 8 is a blank lane.
	Figure 4. ETAA1 can substitute for TopBP1 when present at a sufficiently high concentration. (a) Egg extracts (lane 1) were mock-depleted with control antibodies (lane 2) or immunodepleted with anti-TopBP1 antibodies (lane 3) and immunoblotted with anti-TopBP1 (top) or anti-ETAA1 antibodies (bottom). (b) Mock-depleted (lanes 1 and 2) and TopBP1-depleted extracts (lanes 3–5) were incubated in the absence (lane 1) or presence of APH (lane 3–5). In addition, some extracts were supplemented with recombinant BRCT I-III domain of TopBP1 (lanes 4 and 5) and recombinant HFS-ETAA1 protein (lane 5). Nuclear fractions from the extracts were immunoblotted with the indicated antibodies. (c) Mock-depleted extracts (lanes 1–4) were incubated in the absence (lanes 1 and 3) or presence of APH (lanes 2 and 4). Some extracts were supplemented with recombinant HFS-ETAA1 protein (lanes 3 and 4). Nuclear fractions from the extracts were immunoblotted with the indicated antibodies.
	Figure 5. Full-length ETAA1 activates ATR-ATRIP in a defined biochemical system. (a) Isolation of ATR-ATRIP complexes. Control buffer (lane 1) and full-length ATRIP-FLAG (lane 2) were added to egg extracts containing anti-FLAG M2 antibody beads. After incubation, the beads were reisolated, washed, and incubated with 3X-FLAG peptide. The eluates were immunoblotted with anti-ATR (top) and anti-FLAG antibodies (bottom). (b) Purification of RPA. Recombinant human RPA was purified as described in Materials and Methods and stained with Coomassie blue. (c) Control eluates (lanes 1–5) and eluates containing ATR-ATRIP complex (lanes 6–12) from panel A were incubated in kinase buffer containing γ-[32P]ATP in the absence (lanes 1, 6, and 11) or presence HFS-ETAA1 protein (lanes 2–5, 7–10, and 12). Some incubations also contained RPA (lanes 3, 5, 8, and 10–12), ssDNA (lanes 4–5, and 9–12), and 50 μM ATRi (lane 12). Reactions were subjected to SDS gel electrophoresis. The gel was stained with Coomassie blue (top panel). Incorporation of 32P into proteins was detected by phosphorimaging (second panel from top). For the third panel from the top, we have depicted a longer exposure of the section of the gel containing 32P-labeled RPA32. For the bottom panel, we electrophoresed aliquots of the same samples in a different gel and performed immunoblotting with anti-phospho-RPA32 antibodies.
	Figure 6. ETAA1-dependent phosphorylation of RPA requires an intact AAD. (a) Alignment of AADs around the critical tryptophan residues in ETAA1 and TopBP1 from Xenopus laevis and humans. The conserved tryptophan (W63) in Xenopus ETAA1 is denoted with an asterisk. (b) Wild-type (WT) and mutant W63R HFS-ETAA1 proteins were purified with anti-FLAG antibodies as described in Materials and Methods and stained with Coomassie blue. (c) Control eluates (lanes 1–5) and eluates containing ATR-ATRIP complexes (lanes 6–12) were incubated in kinase buffer containing γ-[32P]ATP in the absence of recombinant HFS-ETAA1 (lanes 1 and 6) or the presence of either WT (lanes 2–3 and 7–9) or W63R HFS-ETAA1 protein (lanes 4–5 and10-12). Some incubations also contained RPA (lanes 3, 5, 8–9, and 11–12) and ssDNA (lanes 3, 5, 9, and 12). Reactions were subjected to SDS gel electrophoresis. The gel was stained with Coomassie blue (top panel). Incorporation of 32P into proteins was detected by phosphorimaging (bottom panel). The portion of the image containing 32P-RPA32 is depicted.
	Figure 7. RPA-ssDNA can still promote activation of a mutant ATR-ATRIP complex that lacks the RPA-binding domain in ATRIP. (a) Control eluates (lanes 1–2) and eluates containing ATR in a complex with either full-length ATRIP (lanes 3–6) or ΔN222 ATRIP (lanes 7–10) were incubated in kinase buffer containing γ-[32P]ATP in the absence (lanes 1, 3, and 7) or presence HFS-ETAA1 protein (lanes 2, 4–6, and 8–10). Some incubations also contained RPA (lanes 5–6 and 9–10) and ssDNA (lanes 6 and 10). Reactions were subjected to SDS gel electrophoresis. The gel was stained with Coomassie blue (top panel). Incorporation of 32P into proteins was detected by phosphorimaging (middle panel). The portion of the image containing 32P-RPA32 is depicted. For the bottom panel, we electrophoresed aliquots of the same samples in a different gel and performed immunoblotting with anti-phospho-RPA32 antibodies. (b) ATR-ATRIP complexes. Full-length (lane 1) and ΔN222 versions of ATRIP-FLAG (lane 2) were added to egg extracts containing anti-FLAG M2 antibody beads. After incubation, the beads were reisolated, washed, and eluted with 3X-FLAG peptide. The eluates were immunoblotted with anti-ATR (top) and anti-ATRIP antibodies (bottom).

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