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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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LAP2β interacts with HA95 via two distinct domains. (A) GST–LAP2β deletion peptides. (B) Indicated peptides were incubated in nuclear extracts of Bjab cells expressing HA95–Myc and sedimented by GST precipitation. Binding of HA95–Myc to peptides was analyzed by immunoblotting using anti-Myc antibodies. Control incubations contained GST or glutathione beads alone. (C) GST–LAP2β(137–373) was incubated in a nuclear extract of Myc-tagged p70S6 kinase-expressing cells, and binding (or lack thereof) of p70S6 kinase to the peptide was detected by GST precipitation and immunoblotting. (D) GST–LAP2β overlays of immunoprecipitated HA95 or B-type lamins. Peptide binding was detected using anti-GST antibodies. (E) Peptide-mediated dissociation of LAP2β from HA95. HA95-IPs were incubated with 100 μM GST–LAP2β peptides, HA95-IPs were sedimented, and pellets and supernatants were immunoblotted using the indicated antibodies. Arrows indicate incubation with LAP2β(137–298) followed by addition of LAP2β(299–373) (lanes 7 and 8) or vice versa (lanes 9 and 10).
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fig2: LAP2β interacts with HA95 via two distinct domains. (A) GST–LAP2β deletion peptides. (B) Indicated peptides were incubated in nuclear extracts of Bjab cells expressing HA95–Myc and sedimented by GST precipitation. Binding of HA95–Myc to peptides was analyzed by immunoblotting using anti-Myc antibodies. Control incubations contained GST or glutathione beads alone. (C) GST–LAP2β(137–373) was incubated in a nuclear extract of Myc-tagged p70S6 kinase-expressing cells, and binding (or lack thereof) of p70S6 kinase to the peptide was detected by GST precipitation and immunoblotting. (D) GST–LAP2β overlays of immunoprecipitated HA95 or B-type lamins. Peptide binding was detected using anti-GST antibodies. (E) Peptide-mediated dissociation of LAP2β from HA95. HA95-IPs were incubated with 100 μM GST–LAP2β peptides, HA95-IPs were sedimented, and pellets and supernatants were immunoblotted using the indicated antibodies. Arrows indicate incubation with LAP2β(137–298) followed by addition of LAP2β(299–373) (lanes 7 and 8) or vice versa (lanes 9 and 10).

Mentions: To map the domains of LAP2β involved in the interaction with HA95, GST–LAP2β fusion polypeptides were produced (Fig. 2 A) (Furukawa et al., 1995, 1997, 1998). Binding of each peptide to HA95–Myc was determined in GST precipitations after incubation of the peptides in a nuclear extract from Bjab cells expressing HA95–Myc. Control extracts were incubated with GST or glutathione beads alone. GST precipitates were immunoblotted using anti-Myc antibodies. Fig. 2 B shows that LAP2β(1–452), (1–397), (1–298), (137–373), (137–298), (243–397), (243–373), (299–397), and (299–373) precipitated HA95–Myc. In contrast, LAP2β(1–193), (1–85), or (194–298) did not precipitate HA95–Myc. Thus, a first HA95-binding domain localizes to amino acids 137–242 of LAP2β and a second domain coincides with the lamin B–binding domain at residues 299–373. We designated these domains HA95-NBD (for HA95 NH2-terminal binding domain) and HA95-CBD (HA95 COOH-terminal binding domain), respectively.


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)

LAP2β interacts with HA95 via two distinct domains. (A) GST–LAP2β deletion peptides. (B) Indicated peptides were incubated in nuclear extracts of Bjab cells expressing HA95–Myc and sedimented by GST precipitation. Binding of HA95–Myc to peptides was analyzed by immunoblotting using anti-Myc antibodies. Control incubations contained GST or glutathione beads alone. (C) GST–LAP2β(137–373) was incubated in a nuclear extract of Myc-tagged p70S6 kinase-expressing cells, and binding (or lack thereof) of p70S6 kinase to the peptide was detected by GST precipitation and immunoblotting. (D) GST–LAP2β overlays of immunoprecipitated HA95 or B-type lamins. Peptide binding was detected using anti-GST antibodies. (E) Peptide-mediated dissociation of LAP2β from HA95. HA95-IPs were incubated with 100 μM GST–LAP2β peptides, HA95-IPs were sedimented, and pellets and supernatants were immunoblotted using the indicated antibodies. Arrows indicate incubation with LAP2β(137–298) followed by addition of LAP2β(299–373) (lanes 7 and 8) or vice versa (lanes 9 and 10).
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Related In: Results  -  Collection

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fig2: LAP2β interacts with HA95 via two distinct domains. (A) GST–LAP2β deletion peptides. (B) Indicated peptides were incubated in nuclear extracts of Bjab cells expressing HA95–Myc and sedimented by GST precipitation. Binding of HA95–Myc to peptides was analyzed by immunoblotting using anti-Myc antibodies. Control incubations contained GST or glutathione beads alone. (C) GST–LAP2β(137–373) was incubated in a nuclear extract of Myc-tagged p70S6 kinase-expressing cells, and binding (or lack thereof) of p70S6 kinase to the peptide was detected by GST precipitation and immunoblotting. (D) GST–LAP2β overlays of immunoprecipitated HA95 or B-type lamins. Peptide binding was detected using anti-GST antibodies. (E) Peptide-mediated dissociation of LAP2β from HA95. HA95-IPs were incubated with 100 μM GST–LAP2β peptides, HA95-IPs were sedimented, and pellets and supernatants were immunoblotted using the indicated antibodies. Arrows indicate incubation with LAP2β(137–298) followed by addition of LAP2β(299–373) (lanes 7 and 8) or vice versa (lanes 9 and 10).
Mentions: To map the domains of LAP2β involved in the interaction with HA95, GST–LAP2β fusion polypeptides were produced (Fig. 2 A) (Furukawa et al., 1995, 1997, 1998). Binding of each peptide to HA95–Myc was determined in GST precipitations after incubation of the peptides in a nuclear extract from Bjab cells expressing HA95–Myc. Control extracts were incubated with GST or glutathione beads alone. GST precipitates were immunoblotted using anti-Myc antibodies. Fig. 2 B shows that LAP2β(1–452), (1–397), (1–298), (137–373), (137–298), (243–397), (243–373), (299–397), and (299–373) precipitated HA95–Myc. In contrast, LAP2β(1–193), (1–85), or (194–298) did not precipitate HA95–Myc. Thus, a first HA95-binding domain localizes to amino acids 137–242 of LAP2β and a second domain coincides with the lamin B–binding domain at residues 299–373. We designated these domains HA95-NBD (for HA95 NH2-terminal binding domain) and HA95-CBD (HA95 COOH-terminal binding domain), respectively.

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