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Related Concept Videos

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An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication...
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Related Experiment Video

Updated: Feb 3, 2026

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins
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Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

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Fork pausing allows centromere DNA loop formation and kinetochore assembly.

Diana M Cook1, Maggie Bennett1, Brandon Friedman1

  • 1Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280.

Proceedings of the National Academy of Sciences of the United States of America
|October 31, 2018
PubMed
Summary

De novo kinetochore assembly requires the COMA complex, but replication fork stalling allows alternative assembly pathways. DNA looping, facilitated by stalled forks, is crucial for kinetochore formation when COMA is absent.

Keywords:
COMAcentromerekinetochore

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Kinetochore assembly is essential for accurate chromosome segregation during cell division.
  • The COMA complex plays a key role in cohesin recruitment and canonical kinetochore assembly.
  • Understanding alternative kinetochore assembly mechanisms is vital for comprehending cell cycle regulation and potential therapeutic targets.

Purpose of the Study:

  • To investigate the role of COMA in de novo kinetochore assembly.
  • To explore how replication fork stalling influences kinetochore formation.
  • To elucidate the mechanism by which DNA looping facilitates kinetochore assembly in COMA mutants.

Main Methods:

  • Utilized phleomycin (PHL) and hydroxyurea to induce replication fork stalling.
  • Investigated de novo kinetochore assembly in COMA mutants under various stalling conditions.
  • Employed DNA looping simulations to analyze the thermodynamics of DNA configurations during replication.

Main Results:

  • De novo kinetochore assembly, unlike template-directed assembly, is COMA-dependent.
  • Replication fork stalling activates de novo kinetochore assembly in COMA mutants.
  • Centromere DNA looping occurs at de novo assembly sites and is favored by stalled replication forks.

Conclusions:

  • Replication fork stalling provides a window for DNA looping, enabling COMA-independent kinetochore assembly.
  • DNA loop formation, driven by thermal fluctuations during stalled replication, is critical for sister centromere separation and kinetochore assembly.
  • This study reveals a novel mechanism for kinetochore assembly, highlighting the importance of replication fork dynamics.