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Characterization of Schizosaccharomyces pombe Hus1: a PCNA-related protein that associates with Rad1 and Rad9

T Caspari1, M Dahlen, G Kanter-Smoler

  • 1MRC Cell Mutation Unit, University of Sussex, Brighton BN1 9RR, United Kingdom.

Molecular and Cellular Biology
|January 29, 2000
PubMed

Insights

Hus1 is a key protein in DNA integrity checkpoints. This study shows Hus1 forms a complex with Rad9 and Rad1, and its nuclear localization depends on Rad17.

Area of Science:

  • Cellular biology
  • Molecular genetics
  • DNA repair mechanisms

Background:

  • Hus1 is a checkpoint Rad protein essential for DNA integrity in Schizosaccharomyces pombe.
  • Understanding Hus1's interactions and localization is crucial for elucidating DNA damage response pathways.

Purpose of the Study:

  • To characterize the different forms of Hus1 and its protein interactions.
  • To investigate the role of Rad17 in Hus1 localization and function within DNA integrity checkpoints.

Main Methods:

  • MYC-tagged Hus1 expression and analysis of its various forms.
  • Co-immunoprecipitation to identify interacting proteins (Rad9, Rad1).
  • Indirect immunofluorescence to determine Hus1 subcellular localization.
  • Protein complex fractionation and two-hybrid assays to study protein interactions.

Main Results:

  • Hus1 exists in at least four forms, with Hus1-B being the predominant form involved in a complex with Rad9 and Rad1.
  • Hus1-B undergoes phosphorylation, increasing after DNA irradiation.
  • Hus1 localizes to the nucleus, a process dependent on Rad17.
  • Rad17 forms a distinct complex separate from Rad1, Rad9, and Hus1, but transient interactions between Rad1 and Rad17 were observed.

Conclusions:

  • Hus1 is a crucial component of the DNA integrity checkpoint, interacting with Rad9 and Rad1.
  • Rad17 plays a vital role in regulating Hus1's nuclear localization.
  • The findings provide insights into the assembly and regulation of DNA checkpoint protein complexes.

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