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

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.DNA damage provokes multiple cellular responses, including the activation of DNA damage checkpoint signaling pathways, which arrest cell cycle progression in G 1 , S, and G 2 /M and possibly coordinate repair of the damage. Loss of the DNA damage checkpoint response leads to increased sensitivity to DNA-damaging agents, demonstrating that these pathways are critical for efficient recovery from DNA damage (reviewed in Refs. 1-6). The components of the checkpoint signaling pathways were initially identified by genetic analyses in the yeasts, Schizosaccharomyces pombe and Saccharomyces cerevisiae, and recent studies demonstrated that the checkpoint genes are conserved between yeasts and humans (7-23). Collectively, these studies have given rise to a conserved model in which DNA damage activates members of the ATM (ataxia telangiectasia-mutated) family of phosphatidylinositol 3-kinase-related kinases. These protein kinases are then required to activate the downstream protein kinases Chk1 and Chk2 (20,(22)(23)(24)(25), which regulate cell cycle arrest and possibly other DNA damage-induced responses.Despite the recent progress in the dissection of the downstream protein kinase-mediated portions of the checkpoint signaling pathways, yeast genetic studies demonstrated that additional checkpoint genes also regulate the DNA damage response (reviewed in Refs. 2-6). For example, in S. pombe spRad1, spHus1, spRad9, and spRad17 (using S. pombe nomenclature) are all essential for DNA damage-induced cell cycle arrest and activation of the spChk1 protein kinase after DNA damage (4 -6). Thus, these results suggest that Rad1, Hus1, Rad9, and Rad17 function upstream of Chk1 activation in the DNA damage-signaling pathw...

Human hRad1 but not hRad9 protects hHus1 from ubiquitin–proteasomal degradation

“…We found that hHus1 was relatively unstable when compared to its close family members hRad1 and hRad9 in cells. The hRad1 protein is a stable molecule and its expression is not affected by hRad9 and hHus1, consistent with the findings that a large proportion of hRad1 can exist as a monomer in cells (Burtelow et al, 2001). The stability of hHus1 expression depends upon the presence of hRad1 but not hRad9, implying that hHus1 and hRad1 may form an ordered complex prior to the association hHus1-hRad9 for the formation of the hRad9-hRad1-hHus1 checkpoint complex.…”

“…It has been reported that the N-terminus of hHus1 interacts with the C-terminus of hRad9 (Burtelow et al, 2001). Therefore, it is possible that the failure of hRad9 to stabilize Myc-hHus1 is due to the c-Myc tag presented in the N-terminus of hHus1 that interferes with the association between hHus1 and hRad9.…”

Regulation and mechanisms of mammalian double-strand break repair

“…Another target for ATM and ATR phosphorylation in S phase is the 9-1-1 complex (Thelen et al, 1999;Burtelow et al, 2001;Lindsey-Boltz et al, 2001). Both the 9-1-1 complex and PIKKs are required for CHK1 activation in fission yeast in response to IR (Brondello et al, 1999).…”

Viral infections and cell cycle G2/M regulation

“…The early genes in the pathway, which include Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1, are thought to sense the DNA damage and lead to phosphorylation of the Chk1 protein [11]. In response to double strand DNA breaks (DSBs) induced by ionizing radiation, for example, Rad17 acts as a checkpoint-specific loading factor, which responds to the DNA damage by loading a 9-1-1 protein complex onto the sites where DNA is damaged [12,13]. The 9-1-1 protein com-plex is also known as the checkpoint clamp complex (CCC), which is composed of Rad1, Rad9 and Hus1 [13].…”