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Related Experiment Videos

Nucleosome positioning at the replication fork.

R Lucchini1, R E Wellinger, J M Sogo

  • 1Institute of Cell Biology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.

The EMBO Journal
|December 18, 2001
PubMed
Summary
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Newly replicated DNA rapidly forms nucleosomes within seconds in yeast. This suggests nucleosome positioning is an early step in chromatin maturation after DNA replication.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Nucleosomes are fundamental units of DNA packaging in eukaryotes.
  • Understanding nucleosome assembly and positioning after DNA replication is crucial for chromatin dynamics.
  • The precise timing and mechanisms of nucleosome positioning on newly synthesized DNA remain incompletely understood.

Purpose of the Study:

  • To investigate the kinetics of nucleosome positioning on daughter DNA strands immediately following replication fork passage.
  • To determine how pre-existing and newly assembled nucleosomes position themselves relative to DNA sequences.
  • To elucidate the role of nucleosome positioning in the early stages of chromatin maturation.

Main Methods:

  • Utilized psoralen cross-linking to stabilize replication intermediates.

Related Experiment Videos

  • Purified cross-linked replication intermediates using preparative two-dimensional agarose gel electrophoresis.
  • Analyzed nucleosome positioning by employing exonuclease digestion and primer extension techniques.

    • Main Results:

    • Nucleosomes were found to position themselves on newly replicated DNA strands shortly after the replication machinery passed.
    • Both pre-existing (randomly segregated) and newly assembled histone octamers rapidly adopted favored positions within seconds.
    • Psoralen intercalation sites indicated specific nucleosome positioning within the Saccharomyces cerevisiae rDNA intergenic spacer.

    Conclusions:

    • Nucleosome positioning on newly replicated DNA is a rapid process, occurring within seconds.
    • This rapid positioning suggests it is an initial and critical step in chromatin maturation.
    • The findings provide insights into the dynamic assembly and organization of chromatin post-replication.