Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation.
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.
Zellner
Elisabeth
E
Institute of Biochemistry, Biocenter at the University of Würzburg, Am Hubland, D-97074 Würzburg, Germany.
Herrmann
Thomas
T
Schulz
Carsten
C
Grummt
Friedrich
F
D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D002454:Cell Differentiation; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004261:DNA Replication; D004275:DNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D020459:Genes, rRNA; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D032446:Myoblasts / Q000166:cytology*; D018741:Replication Origin
D004275:DNA, Ribosomal