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Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy
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DNA replication checkpoint control mediated by the spindle checkpoint protein Mad2p in fission yeast

Izumi Sugimoto1, Hiroshi Murakami, Yuko Tonami

  • 1Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.

The Journal of Biological Chemistry
|September 7, 2004
PubMed

Insights

The DNA replication and spindle checkpoints are linked. Mad2p, a spindle checkpoint protein, is required for the DNA replication checkpoint when Cds1p is compromised, particularly in rad3 mutant cells.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • The interplay between DNA replication and spindle assembly checkpoints in cell cycle regulation remains incompletely understood.
  • Spindle formation typically follows DNA replication in most eukaryotes.
  • Fission yeast mutants rad3 and cds1 exhibit distinct responses to DNA replication inhibition.

Purpose of the Study:

  • To elucidate the relationship between DNA replication and spindle checkpoints.
  • To investigate the role of Mad2p in the DNA replication checkpoint when Cds1p function is impaired.
  • To identify components acting downstream of Rad3p in the DNA replication checkpoint pathway.

Main Methods:

  • Utilized fission yeast mutants (rad3, cds1, mad2) to study cell cycle progression under DNA replication stress (hydroxyurea).
  • Assessed mitotic entry timing and progression in various mutant combinations.
  • Investigated physical interactions between checkpoint proteins using biochemical assays.

Main Results:

  • Inhibition of DNA replication in cds1 mutant cells delays mitotic entry due to Mad2p activation.
  • Simultaneous mutation of cds1 and mad2 (cds1 mad2) restored earlier mitotic entry under hydroxyurea treatment.
  • Overexpression of Mad2p or inactivation of Slp1p rescued checkpoint defects in HU-treated rad3 cells.
  • Rad3p mediates the physical interaction between Mad2p and Slp1p during hydroxyurea treatment.

Conclusions:

  • Mad2p and Slp1p function downstream of Rad3p in the DNA replication checkpoint.
  • Mad2p is essential for the DNA replication checkpoint when Cds1p is compromised.
  • These findings reveal a critical link between the DNA replication and spindle checkpoints involving Mad2p and Slp1p.

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