Limits...
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.

Show MeSH
Nuclear injection of GST–LAP2β(137–298) in G1 inhibits replication. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone together with a 150-kD FITC–dextran to visualize injections, and cells were cultured for 9–10 h with BrdU. Mock (buffer)-injected cells were also cultured with BrdU and 50 μM aphidicolin. BrdU incorporation was detected using TRITC-conjugated anti-BrdU antibodies. DNA was labeled with Hoechst 33342. (B) Percentages of cells labeled with BrdU 9–10 h after peptide injection (n = 45–50/treatment in two replicates). (C) Immunofluorescence analysis of Cdc6 and Orc2p in G1 cells injected as in A. Cells were fixed 2 h after peptide injection. Arrows point to noninjected cells. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172640&req=5

fig8: Nuclear injection of GST–LAP2β(137–298) in G1 inhibits replication. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone together with a 150-kD FITC–dextran to visualize injections, and cells were cultured for 9–10 h with BrdU. Mock (buffer)-injected cells were also cultured with BrdU and 50 μM aphidicolin. BrdU incorporation was detected using TRITC-conjugated anti-BrdU antibodies. DNA was labeled with Hoechst 33342. (B) Percentages of cells labeled with BrdU 9–10 h after peptide injection (n = 45–50/treatment in two replicates). (C) Immunofluorescence analysis of Cdc6 and Orc2p in G1 cells injected as in A. Cells were fixed 2 h after peptide injection. Arrows point to noninjected cells. Bars, 10 μm.

Mentions: The significance of HA95 interaction with HA95-NBD or HA95-CBD was further investigated in vivo by injections of ∼5 nM GST–LAP2β peptides into the nuclei of HeLa cells in early G1 (2 h after release from mitotic arrest). Injections were verified by nuclear retention of a 150-kD FITC–dextran (see below and Fig. 8 A).


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 inhibits replication. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone together with a 150-kD FITC–dextran to visualize injections, and cells were cultured for 9–10 h with BrdU. Mock (buffer)-injected cells were also cultured with BrdU and 50 μM aphidicolin. BrdU incorporation was detected using TRITC-conjugated anti-BrdU antibodies. DNA was labeled with Hoechst 33342. (B) Percentages of cells labeled with BrdU 9–10 h after peptide injection (n = 45–50/treatment in two replicates). (C) Immunofluorescence analysis of Cdc6 and Orc2p in G1 cells injected as in A. Cells were fixed 2 h after peptide injection. Arrows point to noninjected cells. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172640&req=5

fig8: Nuclear injection of GST–LAP2β(137–298) in G1 inhibits replication. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone together with a 150-kD FITC–dextran to visualize injections, and cells were cultured for 9–10 h with BrdU. Mock (buffer)-injected cells were also cultured with BrdU and 50 μM aphidicolin. BrdU incorporation was detected using TRITC-conjugated anti-BrdU antibodies. DNA was labeled with Hoechst 33342. (B) Percentages of cells labeled with BrdU 9–10 h after peptide injection (n = 45–50/treatment in two replicates). (C) Immunofluorescence analysis of Cdc6 and Orc2p in G1 cells injected as in A. Cells were fixed 2 h after peptide injection. Arrows point to noninjected cells. Bars, 10 μm.
Mentions: The significance of HA95 interaction with HA95-NBD or HA95-CBD was further investigated in vivo by injections of ∼5 nM GST–LAP2β peptides into the nuclei of HeLa cells in early G1 (2 h after release from mitotic arrest). Injections were verified by nuclear retention of a 150-kD FITC–dextran (see below and Fig. 8 A).

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