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Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae
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Site-specific DNA binding of the Schizosaccharomyces pombe origin recognition complex is determined by the Orc4

D Kong1, M L DePamphilis

  • 1National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2753, USA.

Molecular and Cellular Biology
|November 2, 2001
PubMed

Insights

The origin recognition complex (ORC) identifies DNA replication origins through its Orc4p subunit, which specifically binds to AT-rich sequences. This Orc4p-driven binding is crucial for ORC

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • DNA replication initiation is a fundamental cellular process.
  • Origin Recognition Complexes (ORCs) are essential for initiating DNA replication.
  • Understanding how ORCs recognize specific DNA sequences at replication origins is critical.

Purpose of the Study:

  • To investigate the mechanism by which ORCs identify replication origins in Schizosaccharomyces pombe.
  • To determine which subunit(s) of the ORC complex are responsible for DNA binding specificity.

Main Methods:

  • Purification of Orc proteins from Schizosaccharomyces pombe.
  • In vitro DNA binding assays using purified Orc proteins and origin DNA sequences.
  • Analysis of protein-DNA interactions and protection patterns.

Main Results:

  • Orc4p alone demonstrated tight and specific binding to key sites within S. pombe replication origins.
  • These specific binding sites were characterized by AT-rich clusters, not alternating AT or GC-rich sequences.
  • The presence of other ORC subunits (ORC-5) did not alter Orc4p's specific binding or protection of origin DNA.
  • ORC-5 alone exhibited weak, non-specific DNA binding; strong binding required Orc4p.
  • The complete six-subunit ORC remained bound to chromatin throughout the cell cycle.

Conclusions:

  • Orc4p is the sole determinant of S. pombe ORC's specific DNA binding to replication origins in vitro.
  • The ORC complex binds to multiple specific sites within replication origins.
  • Orc4p's specific recognition of AT-rich sequences guides ORC to replication origins.

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