TY - JOUR
T1 - Reconstitution and Molecular Analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA Damage Responsive Checkpoint Complex
AU - Burtelow, Matthew A.
AU - Roos-Mattjus, Pia M.K.
AU - Rauen, Matthew
AU - Babendure, Jeremy R.
AU - Karnitz, Larry M.
PY - 2001/7/13
Y1 - 2001/7/13
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0035854804&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035854804&partnerID=8YFLogxK
U2 - 10.1074/jbc.M102946200
DO - 10.1074/jbc.M102946200
M3 - Article
C2 - 11340080
AN - SCOPUS:0035854804
SN - 0021-9258
VL - 276
SP - 25903
EP - 25909
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 28
ER -