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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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Purification and analysis of proteins from murine nuclear extracts. (A) Nuclear extracts from FM3A cells were purified twice on cationic exchange columns and once by gel filtration on a Superdex™200 10/300 GL column, calibrated with thyroglobulin and ferritin. To test for DNA-binding activity aliquots of fractions E4 to E15 were subjected to EMSA with 10 fmol γ32P-ATP 5′ end-labeled fragment B in the presence of 5 mM ATP and 250 ng poly dI+dC. Protein/DNA complexes A and B are indicated. (B) Immunoblot analysis of fractions E5 and E11 with antibodies raised against ORC1, -2, -4 and -5, respectively.
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Figure 3: Purification and analysis of proteins from murine nuclear extracts. (A) Nuclear extracts from FM3A cells were purified twice on cationic exchange columns and once by gel filtration on a Superdex™200 10/300 GL column, calibrated with thyroglobulin and ferritin. To test for DNA-binding activity aliquots of fractions E4 to E15 were subjected to EMSA with 10 fmol γ32P-ATP 5′ end-labeled fragment B in the presence of 5 mM ATP and 250 ng poly dI+dC. Protein/DNA complexes A and B are indicated. (B) Immunoblot analysis of fractions E5 and E11 with antibodies raised against ORC1, -2, -4 and -5, respectively.

Mentions: To analyze the DNA-binding activity, purified proteins of >300 kDa mass after gel filtration were incubated with radiolabeled fragment B and assayed by EMSA (Figure 3A). Proteins of several fractions bind to DNA fragment B, forming two major protein/DNA complexes of distinct electrophoretic mobility, designated complexes A and B, respectively. The fractions exerting the most prominent shifts, i.e. E5 (molecular mass of >663 kDa) and E11 (<440 kDa), were examined by immunoblotting using various antibodies specific for components of the pre-RC (Figure 3B). ORC1, -2, -4 and -5 were confirmed in fraction E5. Fraction E11 contained the same proteins but at a lower level than in fraction E5. By this three-step chromatography, ORC containing protein complexes could be enriched from nuclear extracts. Fractions E5 and E11 comprised highest DNA-binding activity resulting in the formation of complex A and B, respectively. Although the presence of pre-RC components was demonstrated in these protein fractions, the data could not provide conclusive evidence whether the complexes are caused primarily by binding of pre-RC components to fragment B or whether they are due to the interaction with unknown proteins.Figure 3.


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)

Purification and analysis of proteins from murine nuclear extracts. (A) Nuclear extracts from FM3A cells were purified twice on cationic exchange columns and once by gel filtration on a Superdex™200 10/300 GL column, calibrated with thyroglobulin and ferritin. To test for DNA-binding activity aliquots of fractions E4 to E15 were subjected to EMSA with 10 fmol γ32P-ATP 5′ end-labeled fragment B in the presence of 5 mM ATP and 250 ng poly dI+dC. Protein/DNA complexes A and B are indicated. (B) Immunoblot analysis of fractions E5 and E11 with antibodies raised against ORC1, -2, -4 and -5, respectively.
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Figure 3: Purification and analysis of proteins from murine nuclear extracts. (A) Nuclear extracts from FM3A cells were purified twice on cationic exchange columns and once by gel filtration on a Superdex™200 10/300 GL column, calibrated with thyroglobulin and ferritin. To test for DNA-binding activity aliquots of fractions E4 to E15 were subjected to EMSA with 10 fmol γ32P-ATP 5′ end-labeled fragment B in the presence of 5 mM ATP and 250 ng poly dI+dC. Protein/DNA complexes A and B are indicated. (B) Immunoblot analysis of fractions E5 and E11 with antibodies raised against ORC1, -2, -4 and -5, respectively.
Mentions: To analyze the DNA-binding activity, purified proteins of >300 kDa mass after gel filtration were incubated with radiolabeled fragment B and assayed by EMSA (Figure 3A). Proteins of several fractions bind to DNA fragment B, forming two major protein/DNA complexes of distinct electrophoretic mobility, designated complexes A and B, respectively. The fractions exerting the most prominent shifts, i.e. E5 (molecular mass of >663 kDa) and E11 (<440 kDa), were examined by immunoblotting using various antibodies specific for components of the pre-RC (Figure 3B). ORC1, -2, -4 and -5 were confirmed in fraction E5. Fraction E11 contained the same proteins but at a lower level than in fraction E5. By this three-step chromatography, ORC containing protein complexes could be enriched from nuclear extracts. Fractions E5 and E11 comprised highest DNA-binding activity resulting in the formation of complex A and B, respectively. Although the presence of pre-RC components was demonstrated in these protein fractions, the data could not provide conclusive evidence whether the complexes are caused primarily by binding of pre-RC components to fragment B or whether they are due to the interaction with unknown proteins.Figure 3.

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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