Zhu XN , Kim DH , Lin HR , Budhavarapu VN , Rosenbaum HB , Mueller PR , Yew PR .

	Figure 1. Xic2 is ubiquitinated and degraded in a DNA dependent manner. A. Degradation assay. 35S-methionine labeled Xic1, Xic2, and Xic3 were incubated in Xenopus interphase egg extract (Low Speed Supernatant, LSS) in the absence (-) or presence (+) of Xenopus sperm chromatin (XSC) for 0 to 3 hours as indicated. The mean percentage of remaining protein from two independent experiments is shown (% protein remaining) where the zero hour time point was normalized to 100%. Xic1, Xic2, and Xic3 protein bands are marked on the right including ubiquitinated [(UB)n] forms of Xic2. B. Xic2 ubiquitination assay. 35S-methionine labeled Xic2 was incubated in LSS with methyl ubiquitin (3 mg/ml) to stabilize the ubiquitinated species in the absence (-) or presence (+) of XSC for 0 to 3 hours as indicated. Xic2 protein bands are marked on the right including ubiquitinated [(UB)n] forms. C. Nuclei spin down assay. Nuclei spin down assay was employed to separate cytosolic (CYT) and nuclear fractions (NUC) after incubation of [35S]-methionine labeled Xic2 with LSS containing XSC. The input (INPUT) represents 1/15th of the sample before centrifugation and the cytosolic (CYT) represents 1/15th of the cytosolic fraction after centrifugation. Xic2-(UB) n denotes ubiquitinated Xic2. D. Xic2 immunoblot. Left panel: Xenopus LSS or XTC cell extracts were immunoprecipitated (IP) using anti-Xic2 (XIC2) or normal rabbit serum (Mock) antibody and then immunoblotted with anti-Xic2 antibody. Ten percent of the immunoprecipitation reaction was loaded directly (10% INPUT). Right panel: XTC cells were treated with gamma irradiation (IR, 10 Gy) and harvested 4 or 8 hrs following treatment. Lysates were then examined by immunoblotting with anti-Xic2 antibody. For all figures, the molecular weight marker (M) is shown in kilodaltons.
	Figure 2. Xic2 is differentially phosphorylated in the absence and presence of single-stranded DNA. A. Xic2 phosphorylation shift assay. 35S-methionine labeled Xic2 was incubated in interphase egg extract (LSS or HSS) or mitotic extract (Δ90) as indicated in the absence (-) or presence of 80 or 200 units (U) of lambda phosphatase (λ-PPase). B. Xic2 phosphorylation shift and degradation assay. 35S-methionine labeled Xic2 was incubated in LSS with buffer (no DNA), single-stranded ΦX174 DNA (ssDNA, 10 ng/ul), pCS2+ plasmid DNA (dsDNA, 10 ng/ul), or XSC (10 ng/ul) at 23°C. Samples were analyzed by SDS-PAGE at 0-3 hrs. The mean percentage of remaining Xic2 from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. C. Xic2 degradation assay. 35S-methionine labeled Xic1 or Xic2 was incubated in HSS with (+) or without (-) single-stranded DNA (ssDNA) for 0–3 hrs as indicated. Molecular weight markers are shown in kilodaltons. D. Schematic representation of 35S-methionine labeled Xic2 phosphoforms in the absence of extract or DNA (IVT) (left lane), in the presence of LSS with or without XSC (middle lane), or in the presence of LSS or HSS with ssDNA (right lane). Unphosphorylated Xic2 is marked by the blue line (XIC2), Xic2 phosphoform 1 is marked by the purple line and the caret (>), and Xic2 phosphoforms 2 are marked by the pink lines and the asterisks (*). E. Xic2 co-immunoprecipitation with cyclin E. 35S-methionine labeled Xic2 was incubated in HSS without (no DNA) or with (ssDNA) as indicated. Xic2 was co-immunoprecipitated (IP) with anti-cyclin E or control antibody. 10% of the input reaction is shown (10% INPUT). In all figures, “XIC2-P” or the caret (<) and asterisks (*) indicate the phosphoforms of Xic2 and ubiquitinated Xic2 protein bands are indicated as “XIC2-(UB)n”.
	Figure 3. Xic2 is degraded in a PCNA-dependent manner. A. Xic2 degradation assay. 35S-labeled Xic2 was incubated in HSS with (+) or without (-) ssDNA (ΦX174) with buffer (methanol) or aphidicolin (100 ng/ul). The mean percentage of remaining Xic2 from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. B. Left top panel: Sequence alignment of the PCNA binding domain in human p21Cip1, Xenopus p27Xic1, and Xenopus p16Xic2. The arrows indicate the critical PCNA binding amino acids in p21Cip1. The rectangle indicates the PIP box sequences and the circle indicates the residue (F123) mutated to disrupt PCNA binding. Left bottom panel: Xic2 GST pull-down assay. GST, GST-Xic2 wildtype (WT), or GST-Xic2 mutants (S98A, F123A, S131A, or T58A) were bound to beads and incubated with LSS. Bound fractions were analyzed by immunoblotting with α-XCyclin E (top) or α-PCNA antibody (bottom). 20% of the input reaction is shown in the left lane (LSS 2ul). Middle panel: Xic2 degradation assay. 35S-labeled Xic2 wildtype (WT) or F123A were incubated in LSS with (+) or without (-) 10 ng/ul XSC. The mean percentage of remaining Xic2 from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. Right panel: The mean relative ubiquitinated Xic2 WT or F123A from two independent experiments. C. PCNA Depletion and Xic2 degradation. Left panel: PCNA immunoblot of HSS not depleted (NOT DEPL), control-depleted (CTRL DEPL), or PCNA-depleted (PCNA DEPL). Middle panel: 35S-labeled Xic2 in HSS that was not depleted (NOT DEPL), control-depleted (CTRL DEPL), or PCNA-depleted (PCNA DEPL) with (+) or without (-) ΦX174 (ssDNA, 10 ng/ul). Right panel: The mean percentage of Xic2 remaining from two independent experiments is shown. For all figures, the ubiquitinated Xic2 protein bands are indicated as “XIC2-(UB)n”.
	Figure 4. Cdt2 readily promotes Xic2 turnover in the presence of p27 CDK inhibitor. A. GST pull-down assay. GST alone or GST fused to wildtype Xic1 (Xic1WT), I174A mutant of Xic1 (Xic1I174A), Xic2, p21, or p27 (shown in coomassie gel shown in bottom panel) were bound to glutathione sepharose and incubated with in vitro translated 35S-methionine labeled Xenopus Cdt2 (XCdt2). 5% of the input XCdt2 is shown in lane 1 of the top panel (5% Input). B. Xic2 degradation assay. Top panel: 35S-methionine labeled Xic2 was incubated in HSS with (+) or without (-) 10 ng/ul ssDNA (ssDNA) in the presence (+) or absence (-) of p27, unprogrammed reticulocyte lysate (Unprog), or unlabeled in vitro translated Cdt2 (CDT2) for 0 to 3 hrs as indicated. Bottom panel: Graphic representation of Xic2 degradation. 35S-methionine labeled Xic2 was incubated in HSS with 10 ng/ul ssDNA and the percentage of Xic2 remaining was calculated for each sample where the zero hour time point was normalized to 100%. Reactions were supplemented with unprogrammed reticulocyte lysate (Unprog) (7 experiments), unlabeled in vitro translated Cdt2 (Cdt2) (7 experiments), unprogrammed reticulocyte lysate with p27 (Unprog+p27) (4 experiments), unlabeled in vitro translated Cdt2 with p27 (Cdt2+p27) (4 experiments), or unlabeled in vitro translated Xenopus Skp2 (Skp2) (3 experiments). Error bars (Standard error of the mean) are shown. P values were calculated by student t-test comparing each sample with the addition of unprogrammed reticulocyte lysate (Unprog). 1.5 hr p values: Cdt2 (0.000463), Unprog+p27 (0.270), Cdt2+p27 (0.00184), Skp2 (0.702). 3 hr p values: Cdt2 (0.00120), Unprog+p27 (0.0130), Cdt2+p27 (6.56E-05), Skp2 (0.306).
	Figure 5. Phosphorylation of residue S98 by CDK2 stabilizes Xic2. A. Degradation assay. 35S-labeled Xic2 was incubated in HSS with buffer control (DMSO), 1mM roscovitine (ROSC), GST (10uM), or GST-hp27 (10uM) with (+) or without (-) ΦX174 (ssDNA, 10ng/ul). The mean percentage of Xic2 remaining from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. B. Schematic representation of Xic2 and S/T-P consensus sites. C. Xic2 degradation assay. 35S-labeled Xic2 wildtype (WT) or CDK phosphorylation mutants (T58A, S98A, or S131A) were incubated in LSS with (+) or without (-) XSC (10ng/ul). The mean percentage of remaining Xic2 from three independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. D. Xic2 degradation assay. 35S-labeled Xic2 wildtype (WT) or triple-mutant (T58A, S98A, S131A) was incubated in LSS with (+) or without (-) XSC (10ng/ul) for 0 to 3 hrs as indicated. The mean percentage of remaining Xic2 from three independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. E. Xic2 degradation assay. Top panel: 35S-labeled Xic2 wildtype (WT) or glutamic acid phosphomimetic E mutants (S98E, S131E, or S98E/S131E) were incubated in HSS with (+) or without (-) 10 ng/ul ssDNA (ssDNA) for 0 to 3 hrs as indicated. Bottom panel: The percentage of Xic2 remaining was calculated for each sample where the zero hour time point was normalized to 100%. Error bars (Standard error of the mean) are shown. P values were calculated by student t-test comparing each sample with wildtype Xic2 (WT). 1.5 hr p values: S98E (0.115), S131E (0.310), and S98E/S131E (0.015). 3 hr p values: S98E (0.370), S131E (0.603), and S98E/S131E (0.172). For all figures, the ubiquitinated Xic2 protein bands are indicated as “XIC2-(UB)n”.
	Figure 6. Single-stranded DNA-dependent Xic2 phosphorylation at residues S78/S81 is sensitive to caffeine treatment. A. Phosphorylation shift assay. 35S-methionine labeled Chk2 and Xic2 were incubated in HSS with (+) or without (-) annealed oligonucleotides DNA (dsDNA ends, 100 ng/ul) for 0 to 3 hrs as indicated. B. Xic2 degradation assay. 35S-methionine labeled Xic2 was incubated in LSS with 10 ng/ul or 50 ng/ul ΦX174 single-stranded DNA (ssDNA) in the presence of XB- (buffer) or 10 mM caffeine for 0 to 3 hrs as indicated. The mean percentage of remaining Xic2 from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. C. Phosphorylation shift assay. 35S-methionine labeled Xic2 was incubated in LSS or HSS as indicated with ΦX174 (ssDNA), XB- (Buffer), nicked pCS2+ plasmid DNA (nicked dsDNA), XSC, UV-irradiated plasmid DNA (UV DNA), uncut plasmid DNA (Uncut), HindIII linearized plasmid DNA (HindIII), StuI linearized plasmid DNA (StuI), or Asp718 linearized plasmid DNA (Asp718) at 10 ng/ul final DNA concentrations for 0 to 3 hrs as indicated. D. Xic2 degradation assay. Top panel: Schematic representation of Xic2 with potential S/T-P and S/T/-Q phosphorylation sites and the proximal sequences surrounding the S/T-Q sites of Xic2. Bottom panel: 35S-methionine labeled Xic2 wildtype (WT) or mutants (S98A, S78A/S81A, or S78A/S81A/S98A) were incubated in LSS with (+) or without (-) 10 ng/ul ΦX174 single-stranded DNA (ssDNA) for 0 to 3 hrs as indicated. The mean percentage of remaining Xic2 from two independent experiments is shown (% Xic2 remaining) where the zero hour time point was normalized to 100%. In all figures, the caret (<) and asterisks (*) indicate the slower migrating phosphoforms of Xic2 and phosphorylated Xic2 is also indicated as XIC2-P.
	Figure 7. Idealized representation of Xic2 phosphoforms, developmental expression, and cell cycle regulation. A. Schematic representation of 35S-methionine labeled Xic2 wildtype and mutant protein bands or Xic2 under specific conditions (untreated, in extract, with DNA, or with kinase inhibitors) (top panel) and schematic representation of Xic2 phosphorylation sites targeted by CDK2-cyclin and a caffeine-sensitive kinase (bottom panel). Unphosphorylated Xic2 is marked by the blue line, S98 phosphorylated Xic2 is marked by the purple line, and S78/S81 phosphorylated Xic2 bands are marked by the pink lines. B. Top Panel: Schematic representation of Xic1, 2, 3 RNA/protein expression during Xenopus development where the thicker lines represent higher expression, the arrow indicates the timing of gastrulation (stage 12) and the diamond indicates the tailbud stage (30). Bottom panel: Xic1, 2, and 3 are predicted to function at the G1 to S phase transition during development and in a normal somatic cell cycle as shown in the drawing (left). It is predicted that Xic1 and Xic2 are targeted by CRL4Cdt2 and CRL1Skp2 ubiquitin ligases. During a response to DNA damage caused by IR (right), both Xic1 and Xic2 are predicted to be upregulated to halt the cell cycle in G1 during a checkpoint to allow DNA repair to occur.

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