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The highly conserved nuclear lamin Ig-fold binds to PCNA: its role in DNA replication.

Shumaker DK, Solimando L, Sengupta K, Shimi T, Adam SA, Grunwald A, Strelkov SV, Aebi U, Cardoso MC, Goldman RD - J. Cell Biol. (2008)

Bottom Line: Studies of nuclear assembly and DNA replication show that lamins, PCNA, and chromatin are closely associated in situ.This inhibitory effect is significantly diminished in nuclei exposed to the same domain bearing the Ig-fold mutation.These findings also provide insights into the mechanisms underlying the numerous disease-causing mutations located within the lamin Ig-fold.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

ABSTRACT
This study provides insights into the role of nuclear lamins in DNA replication. Our data demonstrate that the Ig-fold motif located in the lamin C terminus binds directly to proliferating cell nuclear antigen (PCNA), the processivity factor necessary for the chain elongation phase of DNA replication. We find that the introduction of a mutation in the Ig-fold, which alters its structure and causes human muscular dystrophy, inhibits PCNA binding. Studies of nuclear assembly and DNA replication show that lamins, PCNA, and chromatin are closely associated in situ. Exposure of replicating nuclei to an excess of the lamin domain containing the Ig-fold inhibits DNA replication in a concentration-dependent fashion. This inhibitory effect is significantly diminished in nuclei exposed to the same domain bearing the Ig-fold mutation. Using the crystal structures of the lamin Ig-fold and PCNA, molecular docking simulations suggest probable interaction sites. These findings also provide insights into the mechanisms underlying the numerous disease-causing mutations located within the lamin Ig-fold.

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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.
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fig1: 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.

Mentions: We reasoned that the non–α-helical C-terminal domain of nuclear lamins contained the binding site for PCNA, as previous studies showed that N-terminal–deleted LA and X. laevis LB3 retained their association with PCNA in situ (Spann et al., 1997; Moir et al., 2000a) and that the binding sites for several known lamin-associated proteins are located in this domain (Zastrow et al., 2004). To test this possibility, we added excess LB3T to X. laevis nuclei engaged in DNA replication. Nuclei were assembled for 60 min and LB3T was added for an additional 30 min. Subsequently, biotin-11-2′-deoxyuridine-5′-triphosphate (bio-11-dUTP) was added for 10 min and the nuclei were processed for immunofluorescence. Nuclei treated with ∼19 μM LB3T showed a reduction in nucleotide incorporation as measured by the fluorescence intensity of Texas red streptavidin (Fig. 1, D–F) relative to controls (Fig. 1, A–C). When nuclei were assembled as above but exposed to bio-11-dUTP for 30 min instead of 10 min, there appeared to be a recovery of DNA replication (unpublished data). This recovery of replication is likely caused by the excess of PCNA and LB3 in the X. laevis extract. Nuclei incubated with LB3T containing the mutation R454W (LB3T RW) displayed a bio-11-dUTP pattern similar to controls (Fig. 1, G–I). This mutation in human LA causes Emery-Dreifuss muscular dystrophy (EDMD; Bonne et al., 1999).


The highly conserved nuclear lamin Ig-fold binds to PCNA: its role in DNA replication.

Shumaker DK, Solimando L, Sengupta K, Shimi T, Adam SA, Grunwald A, Strelkov SV, Aebi U, Cardoso MC, Goldman RD - J. Cell Biol. (2008)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2315674&req=5

fig1: 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.
Mentions: We reasoned that the non–α-helical C-terminal domain of nuclear lamins contained the binding site for PCNA, as previous studies showed that N-terminal–deleted LA and X. laevis LB3 retained their association with PCNA in situ (Spann et al., 1997; Moir et al., 2000a) and that the binding sites for several known lamin-associated proteins are located in this domain (Zastrow et al., 2004). To test this possibility, we added excess LB3T to X. laevis nuclei engaged in DNA replication. Nuclei were assembled for 60 min and LB3T was added for an additional 30 min. Subsequently, biotin-11-2′-deoxyuridine-5′-triphosphate (bio-11-dUTP) was added for 10 min and the nuclei were processed for immunofluorescence. Nuclei treated with ∼19 μM LB3T showed a reduction in nucleotide incorporation as measured by the fluorescence intensity of Texas red streptavidin (Fig. 1, D–F) relative to controls (Fig. 1, A–C). When nuclei were assembled as above but exposed to bio-11-dUTP for 30 min instead of 10 min, there appeared to be a recovery of DNA replication (unpublished data). This recovery of replication is likely caused by the excess of PCNA and LB3 in the X. laevis extract. Nuclei incubated with LB3T containing the mutation R454W (LB3T RW) displayed a bio-11-dUTP pattern similar to controls (Fig. 1, G–I). This mutation in human LA causes Emery-Dreifuss muscular dystrophy (EDMD; Bonne et al., 1999).

Bottom Line: Studies of nuclear assembly and DNA replication show that lamins, PCNA, and chromatin are closely associated in situ.This inhibitory effect is significantly diminished in nuclei exposed to the same domain bearing the Ig-fold mutation.These findings also provide insights into the mechanisms underlying the numerous disease-causing mutations located within the lamin Ig-fold.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

ABSTRACT
This study provides insights into the role of nuclear lamins in DNA replication. Our data demonstrate that the Ig-fold motif located in the lamin C terminus binds directly to proliferating cell nuclear antigen (PCNA), the processivity factor necessary for the chain elongation phase of DNA replication. We find that the introduction of a mutation in the Ig-fold, which alters its structure and causes human muscular dystrophy, inhibits PCNA binding. Studies of nuclear assembly and DNA replication show that lamins, PCNA, and chromatin are closely associated in situ. Exposure of replicating nuclei to an excess of the lamin domain containing the Ig-fold inhibits DNA replication in a concentration-dependent fashion. This inhibitory effect is significantly diminished in nuclei exposed to the same domain bearing the Ig-fold mutation. Using the crystal structures of the lamin Ig-fold and PCNA, molecular docking simulations suggest probable interaction sites. These findings also provide insights into the mechanisms underlying the numerous disease-causing mutations located within the lamin Ig-fold.

Show MeSH
Related in: MedlinePlus