Shumaker DK , Solimando L , Sengupta K , Shimi T , Adam SA , Grunwald A , Strelkov SV , Aebi U , Cardoso MC , Goldman RD .

R01 CA31760 NCI NIH HHS , R01 CA031760 NCI NIH HHS

             protein folding

	Figure 1. Addition of LB3T to assembled X. laevis nuclei inhibits DNA replication. Nuclei were assembled for 60 min followed by the addition of control buffer (A–C), ∼19 μM LB3T (D–F), or ∼19 μM LB3TRW (G–I) for 30 min followed by 5 μM bio-11-dUTP for 10 min. Nuclei were fixed, pelleted onto poly-l-lysine–coated coverslips, and prepared for immunofluorescence using Hoechst 33342, rhodamine avidin, and anti-LB3. Control nuclei continued to grow after addition of the buffer (A–C) and incorporated bio-11-dUTP (B). Using the same image capture settings, there was less incorporation of bio-11-dUTP after the addition of LB3T (E), and the nuclei appeared to be the same size as those in buffer controls at 60 min. In contrast, LB3T RW had a lesser effect on incorporation of bio-11-dUTP (H). Bars, 5 μm. (J) X. laevis nuclei were assembled for 60 min and different amounts of LB3T were added for 30 min followed by a 10-min incubation in 2 μC [32P]α-dCTP. The reactions were stopped by the addition of replication sample buffer for 10 min. The samples were run on 0.8% agarose gels and dried under vacuum, and radioactivity was measured. Under these conditions, there was a concentration-dependent inhibition of DNA replication attributable to LB3T. When ∼11 μM LB3T was added, there was an ∼50% decrease, and with ∼19 μM LB3T, replication decreased by ∼92%. (K) We also tested the effect of LB3T RW and determined that at a concentration of ∼19 μM, DNA replication was reduced by ∼50%. Each experiment was repeated three times and the results were averaged.
	Figure 2. LB3T inhibits the association of PCNA with chromatin.X. laevis nuclei were assembled for 60 min before the addition either in the absence (A–H) or presence (I–P) of ∼19 μM His-tagged LB3T for 30 min. Nuclei were prepared for immunofluorescence using antibodies directed against His6 and PCNA and stained with Hoechst 33342. Control nuclei displayed only background His staining (B, D, F, and H) and a significant amount of PCNA mainly associated with chromatin (C, D, G, and H). In nuclei exposed to LB3T, the His-tagged protein was associated with chromatin (I, J, L–N, and P) and there was a substantial decrease in the amount of PCNA staining (K, L, O, and P). LB3T RW was also associated with chromatin but to a lesser extent than LB3T (Q, R, T–V, and X). Nuclei treated in this manner also had a reduced amount of PCNA associated with chromatin (S, T, W, and X). E–H, M–P, and U–X are 5× views of the boxes in D, L, and T. Bars, 5 μm.
	Figure 3. X. laevis LB3T-Ig binds to PCNA in egg extracts. (A) Diagrammatic comparisons of the tail domains of LB3, LA, and LC. Note that the major conserved region among all three tail domains is the Ig-fold motif, which is ∼80% similar between human LA/C and X. laevis LB3. (B) His-tagged LB3T-Ig was bacterially expressed and purified. The purified protein was separated by SDS-PAGE and stained with Coomassie blue. (C) Purified His-tagged LB3T-Ig was bound to a Hi-Trap chelating column, X. laevis high-speed supernatant was passed over the column, and bound proteins were eluted. The fraction shown was eluted with 600 mM NaCl. The eluted proteins were separated by SDS-PAGE and either stained with Coomassie blue or transferred to nitrocellulose and probed with an antibody directed against PCNA. A band of ∼36 kD seen with Coomassie also reacts with the PCNA antibody.
	Figure 4. The lamin Ig-fold motif binds PCNA. (A) Bacterially expressed and purified PCNA was separated by SDS-PAGE and stained with Coomassie blue (PCNA, molecular weight markers on the left). We always observe that the expressed PCNA appeared as one major and one minor band, both of which immunoblotted with anti-PCNA (see E). (B) LA, lamin A tail (LAT), LB1 tail (LB1T), LC tail (LCT), X. laevis LB3 tail (LB3T), and the Ran-binding domain of importin β (Imp βR) were expressed, purified, bound to protein S–agarose beads, and then mixed with purified PCNA. + lanes show experiments where PCNA was added to S-tagged proteins bound to protein S–agarose beads, whereas − lanes show omitted PCNA. The proteins bound to the S–agarose beads were eluted in SDS sample buffer and separated by SDS-PAGE. Gels were stained with Coomassie blue. The results show that LA, LAT, LB1T, LCT, and LB3T bind to PCNA (arrow). (C) The area identified by the arrow (∼37 kD) in Fig. 4 B was enhanced to more easily identify PCNA bound to LB3T. (D) S-tagged LA-Ig was bound to protein S–agarose beads and incubated with PCNA. The bound proteins were separated by SDS-PAGE, transferred to nitrocellulose, and stained with India ink. The location of LA-Ig and PCNA are shown. (E) After immunoblotting, the nitrocellulose blot seen in F (left) was briefly stained with India ink to show that equal amounts of Imp βR (∼50 kD), LA-Ig, and LA Ig-fold R453W (LA-Ig RW; ∼25 kD) were bound to protein S–agarose beads. (F, left) Purified Imp βR, the LA-Ig, and LA-Ig RW were bound to protein S–agarose beads and mixed with purified PCNA. The bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. The separated proteins were transferred to nitrocellulose and immunoblotted with antibodies directed against PCNA. S-tagged Imp βR was used as a negative control. It was found that the amounts of PCNA bound both to protein S–agarose beads alone (not depicted) and to Imp βR bound to protein S–agarose beads were equivalent (Imp βR +). Therefore, this amount of PCNA binding was considered background. Densitometric analysis showed that after subtracting this background, PCNA binding to LA-Ig RW bound ∼28% less PCNA than wild-type LA-Ig. (F, right) Densitometric analysis of the bound PCNA by immunoblotting seen in F (left). Error bars indicate standard deviation of the mean for six experiments.
	Figure 5. LB3 is present on demembranated sperm chromatin and is closely associated with PCNA during nuclear assembly. Demembranated sperm chromatin was exposed to hS, prepared for immunofluorescence with antibodies directed against LB3 and PCNA, and stained with Hoechst 33342 (A–E). Note that PCNA is absent, whereas LB3 is present and frequently appears to be helically wound around the chromatin (D and E). After the addition of X. laevis interphase extracts to sperm head chromatin for 5 min, LB3 is reorganized into patches and foci associated with chromatin (G, I, and J). In addition, PCNA is now present (H) and closely associated with LB3 (I and J). Enlargements of the regions in the boxes in the merged images (D, DNA and LB3; and I, LB3 and PCNA) are shown (E and J, 5.5× view). Bars, 5 μm. (K) The presence of LB3 and the absence of PCNA on demembranated sperm chromatin is also seen by immunoblotting. Demembranated sperm heads in the amount of 50,000 (lane 1), 100,000 (lane 2), or 200,000 (lane 3) were separated by SDS-PAGE, transferred to nitrocellulose, and probed for the presence of LB3 and PCNA. LB3 is seen in all lanes and is quantified relative to lane 3, but no PCNA could be detected.
	Figure 6. LB3 is closely associated with PCNA and chromatin after nuclear envelope assembly. Nuclei were fixed and prepared for immunofluorescence with Hoechst 33342 and antibodies directed against LB3 and PCNA 60 min after assembly was initiated in X. laevis extracts (A–I). There appears to be substantial overlap of DNA, LB3, and PCNA throughout the nucleus and at the nuclear periphery (A–E). The overlay in D shows LB3 and PCNA, whereas E displays DNA, LB3, and PCNA. The area in the box in E was enlarged (6.3×) to show the details of DNA, LB3, PCNA, and the overlay (F–I). We have also examined nuclei after 120 min of assembly both in the nuclear interior (J–M) and at the nuclear periphery (N–Q). These images are approximately the same magnification as in F–I. Frequently, these nuclei have filamentous chromatin (J), which is closely associated with LB3 and PCNA (K–M). The association between these three components is also observed at the nuclear periphery (N–Q). We have used grayscale images of chromatin in A, F, J, and N and blue in the overlays (E, I, M, and Q) for purposes of improved contrast. Bar, 5 μm.
	Figure 7. Possible Ig-fold–PCNA binding sites based on molecular docking simulations. (A) Results of the analysis using the GRAMM-X software drawn in two orthogonal views. All of the top 10 solutions placed the Ig-fold (one of them is shown in red) in the center of the PCNA ring (blue, yellow, and green). As clearly seen in the bottom image, the center of gravity for the Ig-fold was somewhat off the plane of PCNA and on the opposite side of the interdomain-connecting loop. (B) The ClusPro docking analysis placed LA-Ig at one of three main locations on PCNA: near the interdomain connecting loop (red), between two subunits of PCNA (gray), or the back side of the PCNA homotrimer (dark purple). As seen in the bottom image, the Ig-fold motifs could be docked on either side of the PCNA ring. (C) The top solution of docking the LA-Ig (red) onto a single PCNA molecule (blue). The docking occurs at the intersubunit interface usually observed within the PCNA trimer.

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