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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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Dissociation of HA95 from HA95-NBD in G1 elicits degradation of Cdc6. (A) HA95 (top three panels) or Cdc6 (bottom three panels) was immunoprecipitated from G1- or S-phase HeLa cells, and immune precipitates (P) and supernatants (S) were immunoblotted using either precipitating antibody. (B) Nuclei from G1-phase cells (lane 1) or G1 nuclei loaded with the indicated GST–LAP2β peptides (lanes 2–5) and incubated in nuclear isolation buffer for 1 h were immunoblotted using the indicated antibodies. In lanes 6–8, G1 nuclei loaded with LAP2β(137–298) were incubated in buffer for 1 h together with 25 μM β-lactone, LLnL, or LLM. In lane 9, whole samples (WS; nuclei + buffer) were immunoblotted. (C) G1 nuclei were loaded with GST–LAP2β(137–298) or GST–LAP2β(299–373) in the presence of no inhibitor (−), β-lactone, LLnL, or LLM. Nuclei were allowed to replicate in S-phase extract containing [α32P]dCTP, and synthesized DNA was analyzed by autoradiography.
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fig6: Dissociation of HA95 from HA95-NBD in G1 elicits degradation of Cdc6. (A) HA95 (top three panels) or Cdc6 (bottom three panels) was immunoprecipitated from G1- or S-phase HeLa cells, and immune precipitates (P) and supernatants (S) were immunoblotted using either precipitating antibody. (B) Nuclei from G1-phase cells (lane 1) or G1 nuclei loaded with the indicated GST–LAP2β peptides (lanes 2–5) and incubated in nuclear isolation buffer for 1 h were immunoblotted using the indicated antibodies. In lanes 6–8, G1 nuclei loaded with LAP2β(137–298) were incubated in buffer for 1 h together with 25 μM β-lactone, LLnL, or LLM. In lane 9, whole samples (WS; nuclei + buffer) were immunoblotted. (C) G1 nuclei were loaded with GST–LAP2β(137–298) or GST–LAP2β(299–373) in the presence of no inhibitor (−), β-lactone, LLnL, or LLM. Nuclei were allowed to replicate in S-phase extract containing [α32P]dCTP, and synthesized DNA was analyzed by autoradiography.

Mentions: Initiation of DNA replication requires the assembly of preRCs at origins of replication in G1. The chromatin-bound ORC complex recruits Cdc6, which in turn promotes targeting of MCM proteins. HA95 and Cdc6 were found to coimmunoprecipitate from G1-phase HeLa cells; nevertheless, whereas anti-HA95 antibodies precipitated all detectable Cdc6, a substantial fraction of HA95 did not associate with the Cdc6 immune precipitate (Fig. 6 A). In S phase however, Cdc6 and HA95 did not coprecipitate (Fig. 6 A), despite the reported persistence of a fraction of Cdc6 on chromatin beyond G1 (Mendez and Stillman, 2000).


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)

Dissociation of HA95 from HA95-NBD in G1 elicits degradation of Cdc6. (A) HA95 (top three panels) or Cdc6 (bottom three panels) was immunoprecipitated from G1- or S-phase HeLa cells, and immune precipitates (P) and supernatants (S) were immunoblotted using either precipitating antibody. (B) Nuclei from G1-phase cells (lane 1) or G1 nuclei loaded with the indicated GST–LAP2β peptides (lanes 2–5) and incubated in nuclear isolation buffer for 1 h were immunoblotted using the indicated antibodies. In lanes 6–8, G1 nuclei loaded with LAP2β(137–298) were incubated in buffer for 1 h together with 25 μM β-lactone, LLnL, or LLM. In lane 9, whole samples (WS; nuclei + buffer) were immunoblotted. (C) G1 nuclei were loaded with GST–LAP2β(137–298) or GST–LAP2β(299–373) in the presence of no inhibitor (−), β-lactone, LLnL, or LLM. Nuclei were allowed to replicate in S-phase extract containing [α32P]dCTP, and synthesized DNA was analyzed by autoradiography.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172640&req=5

fig6: Dissociation of HA95 from HA95-NBD in G1 elicits degradation of Cdc6. (A) HA95 (top three panels) or Cdc6 (bottom three panels) was immunoprecipitated from G1- or S-phase HeLa cells, and immune precipitates (P) and supernatants (S) were immunoblotted using either precipitating antibody. (B) Nuclei from G1-phase cells (lane 1) or G1 nuclei loaded with the indicated GST–LAP2β peptides (lanes 2–5) and incubated in nuclear isolation buffer for 1 h were immunoblotted using the indicated antibodies. In lanes 6–8, G1 nuclei loaded with LAP2β(137–298) were incubated in buffer for 1 h together with 25 μM β-lactone, LLnL, or LLM. In lane 9, whole samples (WS; nuclei + buffer) were immunoblotted. (C) G1 nuclei were loaded with GST–LAP2β(137–298) or GST–LAP2β(299–373) in the presence of no inhibitor (−), β-lactone, LLnL, or LLM. Nuclei were allowed to replicate in S-phase extract containing [α32P]dCTP, and synthesized DNA was analyzed by autoradiography.
Mentions: Initiation of DNA replication requires the assembly of preRCs at origins of replication in G1. The chromatin-bound ORC complex recruits Cdc6, which in turn promotes targeting of MCM proteins. HA95 and Cdc6 were found to coimmunoprecipitate from G1-phase HeLa cells; nevertheless, whereas anti-HA95 antibodies precipitated all detectable Cdc6, a substantial fraction of HA95 did not associate with the Cdc6 immune precipitate (Fig. 6 A). In S phase however, Cdc6 and HA95 did not coprecipitate (Fig. 6 A), despite the reported persistence of a fraction of Cdc6 on chromatin beyond G1 (Mendez and Stillman, 2000).

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