Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• The Origin Recognition Complex (ORC) binds replication origins throughout the cell cycle in yeast.
• Conflicting data exist regarding ORC's chromatin binding and removal in animal cells.
• Understanding ORC's cell cycle chromatin association is key to DNA replication control.

Purpose of the Study:

• To investigate the cell-cycle-dependent chromatin binding of ORC subunits in animal cells.
• To determine if ORC is removed from chromatin during specific cell cycle phases.
• To elucidate the in vivo dynamics of ORC-chromatin interactions.

Main Methods:

• Utilized Chinese hamster fibroblasts for cell culture.
• Assessed metabolic stability and chromatin co-fractionation of ORC1, ORC2, and ORC4.
• Employed Fluorescence Loss In Photobleaching (FLIP) for in vivo chromatin interaction studies.

Main Results:

• ORC1, ORC2, and ORC4 are metabolically stable and co-fractionate with chromatin throughout the cell cycle.
• In vivo FLIP studies show ORC1 and ORC4 exhibit dynamic chromatin exchange kinetics.
• ORC's in vivo interaction with chromatin remains stable across the cell cycle, including S-phase.

Conclusions:

• ORC subunits dynamically associate with chromatin throughout the entire cell cycle in animal cells.
• The data support a model of dynamic ORC-chromatin interaction rather than stable binding or complete removal.
• This dynamic interaction is likely essential for regulating DNA replication initiation.