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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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Disruption of HA95 interaction with LAP2β does not interfere with nuclear assembly in vitro. (A) Purified HeLa nuclei (Nuc) were disassembled in mitotic extract, and chromosomes (Chr) were immunoblotted using the indicated antibodies. (B) Indicated GST–LAP2β peptides were allowed to bind to chromosomes, and binding was analyzed by immunofluorescence using anti-GST antibodies. Insets, DNA labeled with Hoescht 33342. (C) Chromosomes harboring the indicated peptides were incubated in interphase extract under conditions promoting nuclear formation. Membrane assembly was examined by DiOC6 labeling and phase contrast microscopy. (D) At the end of incubation, the nuclear distribution of GST–LAP2β peptides, LAP2β, A- and B-type lamins, and BAF was examined by immunofluorescence. (E) Nuclei or chromatin masses were also sedimented through sucrose, and proteins were immunoblotted using the indicated antibodies. Bars, 10 μm.
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fig3: Disruption of HA95 interaction with LAP2β does not interfere with nuclear assembly in vitro. (A) Purified HeLa nuclei (Nuc) were disassembled in mitotic extract, and chromosomes (Chr) were immunoblotted using the indicated antibodies. (B) Indicated GST–LAP2β peptides were allowed to bind to chromosomes, and binding was analyzed by immunofluorescence using anti-GST antibodies. Insets, DNA labeled with Hoescht 33342. (C) Chromosomes harboring the indicated peptides were incubated in interphase extract under conditions promoting nuclear formation. Membrane assembly was examined by DiOC6 labeling and phase contrast microscopy. (D) At the end of incubation, the nuclear distribution of GST–LAP2β peptides, LAP2β, A- and B-type lamins, and BAF was examined by immunofluorescence. (E) Nuclei or chromatin masses were also sedimented through sucrose, and proteins were immunoblotted using the indicated antibodies. Bars, 10 μm.

Mentions: To determine whether interaction between HA95 and LAP2β was involved in NE assembly, we assessed the ability of HA95-binding GST–LAP2β peptides to compete with LAP2β for membrane targeting to chromosomes. Purified HeLa nuclei were disassembled in mitotic extract. The resulting condensed chromosomes contained HA95 and BAF, but no A- or B-type lamins, LAP2β, or lamin B receptor (LBR) (Fig. 3 A). After preincubation with 10 μM of each GST–LAP2β peptide, chromosomes were sedimented and peptide binding was examined by immunofluorescence using anti-GST antibodies. All peptides except GST–LAP2β(194–298) bound chromatin (Fig. 3 B), as anticipated from their ability to bind HA95 (Fig. 2), DNA, or chromatin (see Introduction). Chromosomes were resuspended in interphase extract (Fig. 3 C, Input chrom.) under conditions promoting nuclear assembly. Nuclear morphology was examined by phase contrast microscopy and membrane labeling with DiOC6 after 2 h (Fig. 3 C). Without peptide or with GST alone, >80% of chromatin masses supported nuclear reformation. In contrast, LAP2β fragments (1–452), (1–397), (137–373), (243–397), (299–397), (243–373), and (299–373) inhibited nuclear assembly. Each of these peptides contained the lamin B–binding domain/HA95-CBD. Peptides that do not bind HA95 (nor lamin B) (LAP2β[1–193], [1–85], and [194–298]) did not block membrane assembly, neither did LAP2β(1–298) or (137–298), which both contain HA95-NBD.


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)

Disruption of HA95 interaction with LAP2β does not interfere with nuclear assembly in vitro. (A) Purified HeLa nuclei (Nuc) were disassembled in mitotic extract, and chromosomes (Chr) were immunoblotted using the indicated antibodies. (B) Indicated GST–LAP2β peptides were allowed to bind to chromosomes, and binding was analyzed by immunofluorescence using anti-GST antibodies. Insets, DNA labeled with Hoescht 33342. (C) Chromosomes harboring the indicated peptides were incubated in interphase extract under conditions promoting nuclear formation. Membrane assembly was examined by DiOC6 labeling and phase contrast microscopy. (D) At the end of incubation, the nuclear distribution of GST–LAP2β peptides, LAP2β, A- and B-type lamins, and BAF was examined by immunofluorescence. (E) Nuclei or chromatin masses were also sedimented through sucrose, and proteins were immunoblotted using the indicated antibodies. Bars, 10 μm.
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Related In: Results  -  Collection

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fig3: Disruption of HA95 interaction with LAP2β does not interfere with nuclear assembly in vitro. (A) Purified HeLa nuclei (Nuc) were disassembled in mitotic extract, and chromosomes (Chr) were immunoblotted using the indicated antibodies. (B) Indicated GST–LAP2β peptides were allowed to bind to chromosomes, and binding was analyzed by immunofluorescence using anti-GST antibodies. Insets, DNA labeled with Hoescht 33342. (C) Chromosomes harboring the indicated peptides were incubated in interphase extract under conditions promoting nuclear formation. Membrane assembly was examined by DiOC6 labeling and phase contrast microscopy. (D) At the end of incubation, the nuclear distribution of GST–LAP2β peptides, LAP2β, A- and B-type lamins, and BAF was examined by immunofluorescence. (E) Nuclei or chromatin masses were also sedimented through sucrose, and proteins were immunoblotted using the indicated antibodies. Bars, 10 μm.
Mentions: To determine whether interaction between HA95 and LAP2β was involved in NE assembly, we assessed the ability of HA95-binding GST–LAP2β peptides to compete with LAP2β for membrane targeting to chromosomes. Purified HeLa nuclei were disassembled in mitotic extract. The resulting condensed chromosomes contained HA95 and BAF, but no A- or B-type lamins, LAP2β, or lamin B receptor (LBR) (Fig. 3 A). After preincubation with 10 μM of each GST–LAP2β peptide, chromosomes were sedimented and peptide binding was examined by immunofluorescence using anti-GST antibodies. All peptides except GST–LAP2β(194–298) bound chromatin (Fig. 3 B), as anticipated from their ability to bind HA95 (Fig. 2), DNA, or chromatin (see Introduction). Chromosomes were resuspended in interphase extract (Fig. 3 C, Input chrom.) under conditions promoting nuclear assembly. Nuclear morphology was examined by phase contrast microscopy and membrane labeling with DiOC6 after 2 h (Fig. 3 C). Without peptide or with GST alone, >80% of chromatin masses supported nuclear reformation. In contrast, LAP2β fragments (1–452), (1–397), (137–373), (243–397), (299–397), (243–373), and (299–373) inhibited nuclear assembly. Each of these peptides contained the lamin B–binding domain/HA95-CBD. Peptides that do not bind HA95 (nor lamin B) (LAP2β[1–193], [1–85], and [194–298]) did not block membrane assembly, neither did LAP2β(1–298) or (137–298), which both contain HA95-NBD.

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