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HA95 and LAP2 beta mediate a novel chromatin-nuclear envelope interaction implicated in initiation of DNA replication.

Martins S, Eikvar S, Furukawa K, Collas P - J. Cell Biol. (2003)

Bottom Line: HA95 is a chromatin-associated protein that interfaces the nuclear envelope (NE) and chromatin.Rescue of Cdc6 degradation with proteasome inhibitors restores replication.We propose that an interaction of LAP2beta, or LAP2 proteins, with HA95 is involved in the control of initiation of DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Biochemistry, University of Oslo, Oslo 0317, Norway.

ABSTRACT
HA95 is a chromatin-associated protein that interfaces the nuclear envelope (NE) and chromatin. We report an interaction between HA95 and the inner nuclear membrane protein lamina-associated polypeptide (LAP) 2 beta, and a role of this association in initiation of DNA replication. Precipitation of GST-LAP2 beta fusion proteins and overlays of immobilized HA95 indicate that a first HA95-binding region lies within amino acids 137-242 of LAP2 beta. A second domain sufficient to bind HA95 colocalizes with the lamin B-binding domain of LAP2beta at residues 299-373. HA95-LAP2 beta interaction is not required for NE formation. However, disruption of the association of HA95 with the NH2-terminal HA95-binding domain of LAP2 beta abolishes the initiation, but not elongation, of DNA replication in purified G1 phase nuclei incubated in S-phase extract. Inhibition of replication initiation correlates with proteasome-mediated proteolysis of Cdc6, a component of the prereplication complex. Rescue of Cdc6 degradation with proteasome inhibitors restores replication. We propose that an interaction of LAP2beta, or LAP2 proteins, with HA95 is involved in the control of initiation of DNA replication.

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Characterization of nuclei used in the in vitro DNA replication assay. (A) Nuclei purified from HeLa cells in G1 phase were loaded with GST–LAP2β(1–452). Peptide uptake was analyzed by immunofluorescence using anti-GST antibodies. (B) GST–LAP2β(1–452)-loaded G1 nuclei were exposed to an S-phase extract, and import of Cdc6 was visualized by immunofluorescence. Note that the G1 nuclei contain low detectable levels of Cdc6. (C) Cdc6 import was also examined in nuclei containing the indicated peptides. G1 nuclei were also analyzed by (D) TUNEL and (E) electrophoresis to monitor DNA degradation after exposure to replication extract at 4°C or 37°C. Bars, 10 μm.
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fig4: Characterization of nuclei used in the in vitro DNA replication assay. (A) Nuclei purified from HeLa cells in G1 phase were loaded with GST–LAP2β(1–452). Peptide uptake was analyzed by immunofluorescence using anti-GST antibodies. (B) GST–LAP2β(1–452)-loaded G1 nuclei were exposed to an S-phase extract, and import of Cdc6 was visualized by immunofluorescence. Note that the G1 nuclei contain low detectable levels of Cdc6. (C) Cdc6 import was also examined in nuclei containing the indicated peptides. G1 nuclei were also analyzed by (D) TUNEL and (E) electrophoresis to monitor DNA degradation after exposure to replication extract at 4°C or 37°C. Bars, 10 μm.

Mentions: Nuclei were isolated from G1-phase HeLa cells. GST–LAP2β peptides were introduced into the nuclei after mild treatment with lysolecithin. Lysolecithin was previously shown not to affect dynamic properties of isolated nuclei in in vitro nuclear disassembly assays (Collas et al., 1999). Peptides were taken up by ∼90% of the nuclei, as shown by immunofluorescence using anti-GST antibodies (Fig. 4 A; GST–LAP2β[1–452] is shown). Control and peptide-loaded nuclei were incubated for 3 h in a concentrated (25–30 mg/ml) nuclear and cytosolic extract from S-phase HeLa cells containing [α32P]dCTP, dNTPs, GTP, and an ATP-regenerating system to promote replication. Under these conditions, G1 nuclei loaded with GST–LAP2β(1–452) were capable of importing an exogenous BSA–nuclear localization signal conjugate (unpublished data) or the replication factor Cdc6 (Fig. 4 B). Import was ATP and GTP dependent and blocked by preincubation of the nuclei with antibodies against nucleoporins (Fig. 5 B, mAb414). These results indicate that import took place through nuclear pores rather than passively through a damaged NE, and confirmed a previous report of physiological import of transcription factors by nuclei purified as previously described (Landsverk et al., 2002). We also tested whether peptides containing HA95-NBD or HA95-CBD introduced into G1 nuclei would inhibit nuclear import under the conditions described above, as this would be expected to affect DNA replication. Fig. 4 C shows that none of the peptides distinctly impaired import of Cdc6. Notably, import was permitted by the HA95-NBD–containing peptide LAP2β(137–298) and blocked by mAb414. Lastly, the nuclear DNA did not undergo any detectable degradation upon incubation of the G1 nuclei in the extract at 4°C or 37°C, as judged by TUNEL analysis (Fig. 4 C) and DNA agarose gel electrophoresis (Fig. 4 D). These results indicate that isolated G1 nuclei are functional in import, can be manipulated to introduce peptides, and do not undergo detectable DNA degradation in S-phase extract, and that LAP2β fragments containing either HA95-binding domain do not block nuclear import of Cdc6 in vitro.


HA95 and LAP2 beta mediate a novel chromatin-nuclear envelope interaction implicated in initiation of DNA replication.

Martins S, Eikvar S, Furukawa K, Collas P - J. Cell Biol. (2003)

Characterization of nuclei used in the in vitro DNA replication assay. (A) Nuclei purified from HeLa cells in G1 phase were loaded with GST–LAP2β(1–452). Peptide uptake was analyzed by immunofluorescence using anti-GST antibodies. (B) GST–LAP2β(1–452)-loaded G1 nuclei were exposed to an S-phase extract, and import of Cdc6 was visualized by immunofluorescence. Note that the G1 nuclei contain low detectable levels of Cdc6. (C) Cdc6 import was also examined in nuclei containing the indicated peptides. G1 nuclei were also analyzed by (D) TUNEL and (E) electrophoresis to monitor DNA degradation after exposure to replication extract at 4°C or 37°C. Bars, 10 μm.
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fig4: Characterization of nuclei used in the in vitro DNA replication assay. (A) Nuclei purified from HeLa cells in G1 phase were loaded with GST–LAP2β(1–452). Peptide uptake was analyzed by immunofluorescence using anti-GST antibodies. (B) GST–LAP2β(1–452)-loaded G1 nuclei were exposed to an S-phase extract, and import of Cdc6 was visualized by immunofluorescence. Note that the G1 nuclei contain low detectable levels of Cdc6. (C) Cdc6 import was also examined in nuclei containing the indicated peptides. G1 nuclei were also analyzed by (D) TUNEL and (E) electrophoresis to monitor DNA degradation after exposure to replication extract at 4°C or 37°C. Bars, 10 μm.
Mentions: Nuclei were isolated from G1-phase HeLa cells. GST–LAP2β peptides were introduced into the nuclei after mild treatment with lysolecithin. Lysolecithin was previously shown not to affect dynamic properties of isolated nuclei in in vitro nuclear disassembly assays (Collas et al., 1999). Peptides were taken up by ∼90% of the nuclei, as shown by immunofluorescence using anti-GST antibodies (Fig. 4 A; GST–LAP2β[1–452] is shown). Control and peptide-loaded nuclei were incubated for 3 h in a concentrated (25–30 mg/ml) nuclear and cytosolic extract from S-phase HeLa cells containing [α32P]dCTP, dNTPs, GTP, and an ATP-regenerating system to promote replication. Under these conditions, G1 nuclei loaded with GST–LAP2β(1–452) were capable of importing an exogenous BSA–nuclear localization signal conjugate (unpublished data) or the replication factor Cdc6 (Fig. 4 B). Import was ATP and GTP dependent and blocked by preincubation of the nuclei with antibodies against nucleoporins (Fig. 5 B, mAb414). These results indicate that import took place through nuclear pores rather than passively through a damaged NE, and confirmed a previous report of physiological import of transcription factors by nuclei purified as previously described (Landsverk et al., 2002). We also tested whether peptides containing HA95-NBD or HA95-CBD introduced into G1 nuclei would inhibit nuclear import under the conditions described above, as this would be expected to affect DNA replication. Fig. 4 C shows that none of the peptides distinctly impaired import of Cdc6. Notably, import was permitted by the HA95-NBD–containing peptide LAP2β(137–298) and blocked by mAb414. Lastly, the nuclear DNA did not undergo any detectable degradation upon incubation of the G1 nuclei in the extract at 4°C or 37°C, as judged by TUNEL analysis (Fig. 4 C) and DNA agarose gel electrophoresis (Fig. 4 D). These results indicate that isolated G1 nuclei are functional in import, can be manipulated to introduce peptides, and do not undergo detectable DNA degradation in S-phase extract, and that LAP2β fragments containing either HA95-binding domain do not block nuclear import of Cdc6 in vitro.

Bottom Line: HA95 is a chromatin-associated protein that interfaces the nuclear envelope (NE) and chromatin.Rescue of Cdc6 degradation with proteasome inhibitors restores replication.We propose that an interaction of LAP2beta, or LAP2 proteins, with HA95 is involved in the control of initiation of DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Biochemistry, University of Oslo, Oslo 0317, Norway.

ABSTRACT
HA95 is a chromatin-associated protein that interfaces the nuclear envelope (NE) and chromatin. We report an interaction between HA95 and the inner nuclear membrane protein lamina-associated polypeptide (LAP) 2 beta, and a role of this association in initiation of DNA replication. Precipitation of GST-LAP2 beta fusion proteins and overlays of immobilized HA95 indicate that a first HA95-binding region lies within amino acids 137-242 of LAP2 beta. A second domain sufficient to bind HA95 colocalizes with the lamin B-binding domain of LAP2beta at residues 299-373. HA95-LAP2 beta interaction is not required for NE formation. However, disruption of the association of HA95 with the NH2-terminal HA95-binding domain of LAP2 beta abolishes the initiation, but not elongation, of DNA replication in purified G1 phase nuclei incubated in S-phase extract. Inhibition of replication initiation correlates with proteasome-mediated proteolysis of Cdc6, a component of the prereplication complex. Rescue of Cdc6 degradation with proteasome inhibitors restores replication. We propose that an interaction of LAP2beta, or LAP2 proteins, with HA95 is involved in the control of initiation of DNA replication.

Show MeSH
Related in: MedlinePlus