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Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation.

Zellner E, Herrmann T, Schulz C, Grummt F - Nucleic Acids Res. (2007)

Bottom Line: Nucleotide substitutions revealed that two 9 bp sequence elements, CTCGGGAGA, are essential for the binding of pre-RC proteins to origin DNA within the murine rDNA locus.During myogenic differentiation of C2C12 cells, we demonstrated a reduction of ORC1 and ORC2 by immunoblot analyses.ChIP analyses revealed that ORC1 completely disappears from chromatin of terminally differentiated myotubes, whereas ORC2, -4 and -5 still remain associated.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Biocenter at the University of Würzburg, Am Hubland, D-97074 Würzburg, Germany.

ABSTRACT
Eukaryotic DNA replication initiates at origins of replication by the assembly of the highly conserved pre-replicative complex (pre-RC). However, exact sequences for pre-RC binding still remain unknown. By chromatin immunoprecipitation we identified in vivo a pre-RC-binding site within the origin of bidirectional replication in the murine rDNA locus. At this sequence, ORC1, -2, -4 and -5 are bound in G1 phase and at the G1/S transition. During S phase, ORC1 is released. An ATP-dependent and site-specific assembly of the pre-RC at origin DNA was demonstrated in vitro using partially purified murine pre-RC proteins in electrophoretic mobility shift assays. By deletion experiments the sequence required for pre-RC binding was confined to 119 bp. Nucleotide substitutions revealed that two 9 bp sequence elements, CTCGGGAGA, are essential for the binding of pre-RC proteins to origin DNA within the murine rDNA locus. During myogenic differentiation of C2C12 cells, we demonstrated a reduction of ORC1 and ORC2 by immunoblot analyses. ChIP analyses revealed that ORC1 completely disappears from chromatin of terminally differentiated myotubes, whereas ORC2, -4 and -5 still remain associated.

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DNA/protein complex A binds ATP dependently and sequence selectively to fragment B and is supershifted with antibodies against ORC2. (A) EMSAs were performed with proteins of fractions E5 or E11 (300 ng) and γ32P-ATP 5′ end-labeled fragment B with increasing concentrations of ATP (0.5, 1, 2.5 or 5 mM). (B) For supershifting, binding reactions were performed by incubating γ32P-ATP 5′ end-labeled fragment B and proteins of fraction E5 in the presence of 5 mM ATP with increasing amounts of antibodies against ORC2 as indicated. (C) EMSAs were performed with proteins of fractions E5 or E11 and fragments A, C and D in the absence or presence of 5 mM ATP.
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Figure 4: DNA/protein complex A binds ATP dependently and sequence selectively to fragment B and is supershifted with antibodies against ORC2. (A) EMSAs were performed with proteins of fractions E5 or E11 (300 ng) and γ32P-ATP 5′ end-labeled fragment B with increasing concentrations of ATP (0.5, 1, 2.5 or 5 mM). (B) For supershifting, binding reactions were performed by incubating γ32P-ATP 5′ end-labeled fragment B and proteins of fraction E5 in the presence of 5 mM ATP with increasing amounts of antibodies against ORC2 as indicated. (C) EMSAs were performed with proteins of fractions E5 or E11 and fragments A, C and D in the absence or presence of 5 mM ATP.

Mentions: Next, the effect of ATP on the formation of complexes A and B was examined. The presence of two ATP-binding subunits in eukaryotic ORCs (27,34–36) lead us to expect an ATP-dependent increase in DNA binding if complex A or B contain these pre-RC components. The addition of increasing amounts of ATP to fraction E5 results in a dose-dependent increase of the formation of complex A, not however, on the formation of complex B (Figure 4A). This indicates that complex A, but not complex B, is probably caused by the binding of pre-RC proteins to origin DNA.Figure 4.


Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation.

Zellner E, Herrmann T, Schulz C, Grummt F - Nucleic Acids Res. (2007)

DNA/protein complex A binds ATP dependently and sequence selectively to fragment B and is supershifted with antibodies against ORC2. (A) EMSAs were performed with proteins of fractions E5 or E11 (300 ng) and γ32P-ATP 5′ end-labeled fragment B with increasing concentrations of ATP (0.5, 1, 2.5 or 5 mM). (B) For supershifting, binding reactions were performed by incubating γ32P-ATP 5′ end-labeled fragment B and proteins of fraction E5 in the presence of 5 mM ATP with increasing amounts of antibodies against ORC2 as indicated. (C) EMSAs were performed with proteins of fractions E5 or E11 and fragments A, C and D in the absence or presence of 5 mM ATP.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2175324&req=5

Figure 4: DNA/protein complex A binds ATP dependently and sequence selectively to fragment B and is supershifted with antibodies against ORC2. (A) EMSAs were performed with proteins of fractions E5 or E11 (300 ng) and γ32P-ATP 5′ end-labeled fragment B with increasing concentrations of ATP (0.5, 1, 2.5 or 5 mM). (B) For supershifting, binding reactions were performed by incubating γ32P-ATP 5′ end-labeled fragment B and proteins of fraction E5 in the presence of 5 mM ATP with increasing amounts of antibodies against ORC2 as indicated. (C) EMSAs were performed with proteins of fractions E5 or E11 and fragments A, C and D in the absence or presence of 5 mM ATP.
Mentions: Next, the effect of ATP on the formation of complexes A and B was examined. The presence of two ATP-binding subunits in eukaryotic ORCs (27,34–36) lead us to expect an ATP-dependent increase in DNA binding if complex A or B contain these pre-RC components. The addition of increasing amounts of ATP to fraction E5 results in a dose-dependent increase of the formation of complex A, not however, on the formation of complex B (Figure 4A). This indicates that complex A, but not complex B, is probably caused by the binding of pre-RC proteins to origin DNA.Figure 4.

Bottom Line: Nucleotide substitutions revealed that two 9 bp sequence elements, CTCGGGAGA, are essential for the binding of pre-RC proteins to origin DNA within the murine rDNA locus.During myogenic differentiation of C2C12 cells, we demonstrated a reduction of ORC1 and ORC2 by immunoblot analyses.ChIP analyses revealed that ORC1 completely disappears from chromatin of terminally differentiated myotubes, whereas ORC2, -4 and -5 still remain associated.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Biocenter at the University of Würzburg, Am Hubland, D-97074 Würzburg, Germany.

ABSTRACT
Eukaryotic DNA replication initiates at origins of replication by the assembly of the highly conserved pre-replicative complex (pre-RC). However, exact sequences for pre-RC binding still remain unknown. By chromatin immunoprecipitation we identified in vivo a pre-RC-binding site within the origin of bidirectional replication in the murine rDNA locus. At this sequence, ORC1, -2, -4 and -5 are bound in G1 phase and at the G1/S transition. During S phase, ORC1 is released. An ATP-dependent and site-specific assembly of the pre-RC at origin DNA was demonstrated in vitro using partially purified murine pre-RC proteins in electrophoretic mobility shift assays. By deletion experiments the sequence required for pre-RC binding was confined to 119 bp. Nucleotide substitutions revealed that two 9 bp sequence elements, CTCGGGAGA, are essential for the binding of pre-RC proteins to origin DNA within the murine rDNA locus. During myogenic differentiation of C2C12 cells, we demonstrated a reduction of ORC1 and ORC2 by immunoblot analyses. ChIP analyses revealed that ORC1 completely disappears from chromatin of terminally differentiated myotubes, whereas ORC2, -4 and -5 still remain associated.

Show MeSH
Related in: MedlinePlus