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Related Experiment Videos

Architecture of the human origin recognition complex.

S K Dhar1, L Delmolino, A Dutta

  • 1Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

The Journal of Biological Chemistry
|June 8, 2001
PubMed
Summary
This summary is machine-generated.

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Human origin recognition complex (ORC) subunits ORC2-5 form a stable core. A specific ORC3 fragment inhibits ORC2-3 interaction and blocks cell cycle G(1)-S transition, revealing a key role for ORC in mammalian DNA replication.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Human origin recognition complex (ORC) is essential for DNA replication initiation.
  • Previous studies reported human ORC subunits but lacked detailed characterization of their interactions and functions.

Purpose of the Study:

  • To characterize the subunit composition and interactions within the human ORC.
  • To investigate the role of specific human ORC subunits in cell cycle regulation.

Main Methods:

  • Recombinant expression of human ORC subunits in insect cells using baculovirus system.
  • Protein purification and complex formation analysis.
  • In vitro interaction studies using ORC subunit fragments.
  • Cellular overexpression studies in U2OS cells.

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Main Results:

  • Human ORC2, -3, -4, and -5 form a stable core complex, while ORC1 and ORC6 interact more transiently.
  • Direct interaction between the C-terminal region of ORC2 and the N-terminal region of ORC3 was identified.
  • The N-terminal 200 residues of ORC3 (ORC3N) competitively inhibited ORC2-ORC3 interaction.
  • Overexpression of ORC3N in U2OS cells led to a G(1) cell cycle block.

Conclusions:

  • Human ORC2-5 constitutes a stable core complex with labile interactions for ORC1 and ORC6.
  • Specific interactions between ORC subunits are crucial for ORC complex assembly.
  • The ORC3 subunit plays a critical role in mediating ORC complex formation and is essential for the G(1)-S transition in mammalian cells.