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Related Experiment Videos

Visualization of replication sites in unfixed human cells

A B Hassan1, P R Cook

  • 1Sir William Dunn School of Pathology, University of Oxford, UK.

Journal of Cell Science
|June 1, 1993
PubMed
Summary

DNA replication occurs in concentrated foci within cell nuclei. These replication sites remain stable even after removing most chromatin, suggesting attachment to an underlying nuclear structure.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • DNA replication is essential for cell division and occurs at specific sites within the nucleus.
  • The organization of these replication sites suggests an underlying nuclear scaffold or structure.
  • Conventional fixation methods can alter the distribution of cellular components, potentially affecting the observation of replication sites.

Purpose of the Study:

  • To investigate the in vivo distribution of DNA replication sites in unfixed cells.
  • To determine if nuclear structures organize DNA replication activity.
  • To assess the stability of replication foci and their association with nuclear architecture.

Main Methods:

  • Encapsulation of HeLa cells in agarose microbeads for in situ analysis.
  • Permeabilization in a physiological buffer to maintain cellular integrity.
  • Direct labeling of active replication sites using fluorochrome-conjugated dUTP.
  • Microscopy (conventional and digital fluorescence) to visualize and quantify replication foci.

Main Results:

  • 80-250 distinct DNA replication foci were observed in unfixed HeLa cells.
  • Replication foci remained stable and retained most in vivo activity, refuting aggregation artifacts.
  • Foci demonstrated stability even after removal of approximately 90% of chromatin.
  • Common fixation methods were shown to disrupt the periphery but not the core of these foci.

Conclusions:

  • DNA replication is organized into discrete, stable foci within the cell nucleus.
  • These replication foci are likely attached to a stable underlying nuclear structure.
  • The observed organization is intrinsic and not an artifact of fixation or polymerase aggregation.

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