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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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HA95 coprecipitates with LAP2β in interphase. (A) HA95 or LAP2β was immunoprecipitated (IP) from interphase and mitotic Bjab cells. Precipitates (P) and supernatants (S) were immunoblotted using anti-LAP2β or anti-HA95 antibodies. (B) Myc-tagged HA95 was immunoprecipitated from Bjab cells stably expressing HA95–Myc using anti-Myc antibodies, and precipitates were immunoblotted using indicated antibodies. Control immunoprecipitations were done with preimmune rabbit IgGs. (C) HA95-IPs were extracted with 0, 0.25, 0.5, 0.75, 1.0, or 1.25 M NaCl before sedimentation and immunoblotting of pellets and supernatants.
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fig1: HA95 coprecipitates with LAP2β in interphase. (A) HA95 or LAP2β was immunoprecipitated (IP) from interphase and mitotic Bjab cells. Precipitates (P) and supernatants (S) were immunoblotted using anti-LAP2β or anti-HA95 antibodies. (B) Myc-tagged HA95 was immunoprecipitated from Bjab cells stably expressing HA95–Myc using anti-Myc antibodies, and precipitates were immunoblotted using indicated antibodies. Control immunoprecipitations were done with preimmune rabbit IgGs. (C) HA95-IPs were extracted with 0, 0.25, 0.5, 0.75, 1.0, or 1.25 M NaCl before sedimentation and immunoblotting of pellets and supernatants.

Mentions: HA95 coprecipitates with a protein complex containing LAP2β from the transformed human B cell line Bjab in interphase (Martins et al., 2000). We now show that in Bjab cells, HA95 and LAP2β coprecipitated in interphase but not at mitosis, regardless of the precipitating antibody (Fig. 1 A). Immunoblots of the nonprecipitated (S) fractions indicate that in interphase, HA95 coprecipitated all detectable LAP2β, whereas LAP2β did not precipitate all HA95 (Fig. 1 A). Immunoprecipitation of an HA95–Myc fusion protein stably expressed in Bjab cells using an anti-Myc antibody also coprecipitated LAP2β in interphase but not at mitosis (Fig. 1 B). These results suggest a cell cycle–dependent interaction of HA95 with LAP2β; however, not all HA95 resides in a complex with LAP2β.


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)

HA95 coprecipitates with LAP2β in interphase. (A) HA95 or LAP2β was immunoprecipitated (IP) from interphase and mitotic Bjab cells. Precipitates (P) and supernatants (S) were immunoblotted using anti-LAP2β or anti-HA95 antibodies. (B) Myc-tagged HA95 was immunoprecipitated from Bjab cells stably expressing HA95–Myc using anti-Myc antibodies, and precipitates were immunoblotted using indicated antibodies. Control immunoprecipitations were done with preimmune rabbit IgGs. (C) HA95-IPs were extracted with 0, 0.25, 0.5, 0.75, 1.0, or 1.25 M NaCl before sedimentation and immunoblotting of pellets and supernatants.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172640&req=5

fig1: HA95 coprecipitates with LAP2β in interphase. (A) HA95 or LAP2β was immunoprecipitated (IP) from interphase and mitotic Bjab cells. Precipitates (P) and supernatants (S) were immunoblotted using anti-LAP2β or anti-HA95 antibodies. (B) Myc-tagged HA95 was immunoprecipitated from Bjab cells stably expressing HA95–Myc using anti-Myc antibodies, and precipitates were immunoblotted using indicated antibodies. Control immunoprecipitations were done with preimmune rabbit IgGs. (C) HA95-IPs were extracted with 0, 0.25, 0.5, 0.75, 1.0, or 1.25 M NaCl before sedimentation and immunoblotting of pellets and supernatants.
Mentions: HA95 coprecipitates with a protein complex containing LAP2β from the transformed human B cell line Bjab in interphase (Martins et al., 2000). We now show that in Bjab cells, HA95 and LAP2β coprecipitated in interphase but not at mitosis, regardless of the precipitating antibody (Fig. 1 A). Immunoblots of the nonprecipitated (S) fractions indicate that in interphase, HA95 coprecipitated all detectable LAP2β, whereas LAP2β did not precipitate all HA95 (Fig. 1 A). Immunoprecipitation of an HA95–Myc fusion protein stably expressed in Bjab cells using an anti-Myc antibody also coprecipitated LAP2β in interphase but not at mitosis (Fig. 1 B). These results suggest a cell cycle–dependent interaction of HA95 with LAP2β; however, not all HA95 resides in a complex with LAP2β.

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