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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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Nuclear injection of GST–LAP2β(137–298) in G1 does not alter distribution of NE proteins or BAF. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone, cultured for 2–3 h, and analyzed by immunofluorescence using anti-GST, LAP2β, lamin B (goat polyclonal), or BAF antibodies. Arrows point to noninjected cells. Bar, 10 μm.
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fig7: Nuclear injection of GST–LAP2β(137–298) in G1 does not alter distribution of NE proteins or BAF. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone, cultured for 2–3 h, and analyzed by immunofluorescence using anti-GST, LAP2β, lamin B (goat polyclonal), or BAF antibodies. Arrows point to noninjected cells. Bar, 10 μm.

Mentions: We first assessed whether the distribution of INM and lamina proteins was altered in the injected nuclei. As seen earlier in in vitro–reconstituted nuclei, GST–LAP2β peptides were detected throughout the nucleus for the most part, with a propensity of the anti-GST antibody to decorate the nuclear periphery more strongly (Fig. 7, GST). This, however, was not specific for the peptide injected (Fig. 7, bottom three rows). Immunofluorescence analysis of peptide- and mock (buffer)-injected cells indicated that LAP2β and B-type lamins remained localized at the NE 2–3 h after injection with either peptide (Fig. 7). Similar results were obtained for LBR, emerin, and A-type lamins (unpublished data). Additionally, no alteration in the localization of BAF in peptide-injected and control cells was detected (Fig. 7). BAF remained distributed throughout the nucleoplasm with an enrichment around the periphery. Thus, we could not attribute a noticeable effect of intranuclear peptide injection in G1 on overall nuclear architecture.


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)

Nuclear injection of GST–LAP2β(137–298) in G1 does not alter distribution of NE proteins or BAF. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone, cultured for 2–3 h, and analyzed by immunofluorescence using anti-GST, LAP2β, lamin B (goat polyclonal), or BAF antibodies. Arrows point to noninjected cells. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172640&req=5

fig7: Nuclear injection of GST–LAP2β(137–298) in G1 does not alter distribution of NE proteins or BAF. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone, cultured for 2–3 h, and analyzed by immunofluorescence using anti-GST, LAP2β, lamin B (goat polyclonal), or BAF antibodies. Arrows point to noninjected cells. Bar, 10 μm.
Mentions: We first assessed whether the distribution of INM and lamina proteins was altered in the injected nuclei. As seen earlier in in vitro–reconstituted nuclei, GST–LAP2β peptides were detected throughout the nucleus for the most part, with a propensity of the anti-GST antibody to decorate the nuclear periphery more strongly (Fig. 7, GST). This, however, was not specific for the peptide injected (Fig. 7, bottom three rows). Immunofluorescence analysis of peptide- and mock (buffer)-injected cells indicated that LAP2β and B-type lamins remained localized at the NE 2–3 h after injection with either peptide (Fig. 7). Similar results were obtained for LBR, emerin, and A-type lamins (unpublished data). Additionally, no alteration in the localization of BAF in peptide-injected and control cells was detected (Fig. 7). BAF remained distributed throughout the nucleoplasm with an enrichment around the periphery. Thus, we could not attribute a noticeable effect of intranuclear peptide injection in G1 on overall nuclear architecture.

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