Cargando…
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...
Autores principales: | , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2007
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2175324/ https://www.ncbi.nlm.nih.gov/pubmed/17916579 http://dx.doi.org/10.1093/nar/gkm555 |
_version_ | 1782145461016068096 |
---|---|
author | Zellner, Elisabeth Herrmann, Thomas Schulz, Carsten Grummt, Friedrich |
author_facet | Zellner, Elisabeth Herrmann, Thomas Schulz, Carsten Grummt, Friedrich |
author_sort | Zellner, Elisabeth |
collection | PubMed |
description | 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. |
format | Text |
id | pubmed-2175324 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-21753242008-01-07 Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation Zellner, Elisabeth Herrmann, Thomas Schulz, Carsten Grummt, Friedrich Nucleic Acids Res Molecular Biology 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. Oxford University Press 2007-11 2007-10-04 /pmc/articles/PMC2175324/ /pubmed/17916579 http://dx.doi.org/10.1093/nar/gkm555 Text en © 2007 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Molecular Biology Zellner, Elisabeth Herrmann, Thomas Schulz, Carsten Grummt, Friedrich Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation |
title | Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation |
title_full | Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation |
title_fullStr | Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation |
title_full_unstemmed | Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation |
title_short | Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation |
title_sort | site-specific interaction of the murine pre-replicative complex with origin dna: assembly and disassembly during cell cycle transit and differentiation |
topic | Molecular Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2175324/ https://www.ncbi.nlm.nih.gov/pubmed/17916579 http://dx.doi.org/10.1093/nar/gkm555 |
work_keys_str_mv | AT zellnerelisabeth sitespecificinteractionofthemurineprereplicativecomplexwithorigindnaassemblyanddisassemblyduringcellcycletransitanddifferentiation AT herrmannthomas sitespecificinteractionofthemurineprereplicativecomplexwithorigindnaassemblyanddisassemblyduringcellcycletransitanddifferentiation AT schulzcarsten sitespecificinteractionofthemurineprereplicativecomplexwithorigindnaassemblyanddisassemblyduringcellcycletransitanddifferentiation AT grummtfriedrich sitespecificinteractionofthemurineprereplicativecomplexwithorigindnaassemblyanddisassemblyduringcellcycletransitanddifferentiation |