Reconstitution and Molecular Analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA Damage Responsive Checkpoint Complex

Matthew A. Burtelow, Pia M.K. Roos-Mattjus, Matthew Rauen, Jeremy Babendure, Larry M. Karnitz

Research output: Contribution to journalArticle

107 Citations (Scopus)

Abstract

DNA damage activates cell cycle checkpoint signaling pathways that coordinate cell cycle arrest and DNA repair. Three of the proteins involved in checkpoint signaling, Rad1, Hus1, and Rad9, have been shown to interact by immunoprecipitation and yeast two-hybrid studies. However, it is not known how these proteins interact and assemble into a complex. In the present study we demonstrated that in human cells all the hRad9 and hHus1 and approximately one-half of the cellular pool of hRad1 interacted as a stable, biochemically discrete complex, with an apparent molecular mass of 160 kDa. This complex was reconstituted by co-expression of all three recombinant proteins in a heterologous system, and the reconstituted complex exhibited identical chromatographic behavior as the endogenous complex. Interaction studies using differentially tagged proteins demonstrated that the proteins did not self-multimerize. Rather, each protein had a binding site for the other two partners, with the N terminus of hRad9 interacting with hRad1, the N terminus of hRad1 interacting with hHus1, and the N terminus of hHus1 interacting with the C terminus of hRad9's predicted PCNA-like region. Collectively, these analyses suggest a model of how these three proteins assemble to form a functional checkpoint complex, which we dubbed the 9-1-1 complex.

Original languageEnglish (US)
Pages (from-to)25903-25909
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number28
DOIs
StatePublished - Jul 13 2001
Externally publishedYes

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DNA Damage
DNA
Proteins
Cells
Cell Cycle Checkpoints
Proliferating Cell Nuclear Antigen
Molecular mass
Immunoprecipitation
Recombinant Proteins
DNA Repair
Yeast
Repair
Yeasts
Binding Sites

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Reconstitution and Molecular Analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA Damage Responsive Checkpoint Complex. / Burtelow, Matthew A.; Roos-Mattjus, Pia M.K.; Rauen, Matthew; Babendure, Jeremy; Karnitz, Larry M.

In: Journal of Biological Chemistry, Vol. 276, No. 28, 13.07.2001, p. 25903-25909.

Research output: Contribution to journalArticle

Burtelow, Matthew A. ; Roos-Mattjus, Pia M.K. ; Rauen, Matthew ; Babendure, Jeremy ; Karnitz, Larry M. / Reconstitution and Molecular Analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA Damage Responsive Checkpoint Complex. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 28. pp. 25903-25909.
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