Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle.
In yeast, the Origin Recognition Complex (ORC) is bound to replication origins throughout the cell-cycle, but in animal cells, there are conflicting data as to whether and when ORC is removed from chromatin. We find ORC1, 2 and ORC4 to be metabolically stable proteins that co-fractionate with chromatin throughout the cell-cycle in Chinese hamster fibroblasts. Since cellular extraction methods cannot directly examine the chromatin binding properties of proteins in vivo, we examined ORC:chromatin interactions in living cells. Fluorescence loss in photobleaching (FLIP) studies revealed ORC1 and ORC4 to be highly dynamic proteins during the cell-cycle with exchange kinetics similar to other regulatory chromatin proteins. In vivo interaction with chromatin was not significantly altered throughout the cell-cycle, including S-phase. These data support a model in which ORC subunits dynamically interact with chromatin throughout the cell-cycle.
McNairn
Adrian J
AJ
Department of Biochemistry and Molecular Biology, S.U.N.Y. Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
Okuno
Yukiko
Y
Misteli
Tom
T
Gilbert
David M
DM
D000818:Animals; D016466:CHO Cells; D002453:Cell Cycle / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D006224:Cricetinae; D004261:DNA Replication / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051738:Origin Recognition Complex; D038761:Photobleaching; D021122:Protein Subunits / Q000378:metabolism*; D016196:S Phase / Q000502:physiology
D002843:Chromatin; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D021122:Protein Subunits