Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage

Abstract

The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints. Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p. Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+. Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation. Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes. Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes.

Human Homologs of Schizosaccharomyces pombe Rad1, Hus1, and Rad9 Form a DNA Damage-responsive Protein Complex

“…hRad1, hRad9, and hHus1 Associate in a Checkpoint Complex-Comparisons of the predicted human and yeast protein sequences indicate that the human proteins are 25-30% identical and 53-57% similar to their respective homologs, with homologies extending over extensive portions of each protein (12)(13)(14)(15)(16)(17)(18). In S. pombe, spRad1 and spHus1 associate in wildtype, but not rad9, mutant yeast.…”

“…In S. pombe, spRad1 and spHus1 associate in wildtype, but not rad9, mutant yeast. One interpretation of this result is that spRad9 may physically link spRad1 to spHus1, although this hypothesis has not been validated experimentally (18). To address whether conservation extends to a functional level, we examined the ability of the human homologs to form biochemical complexes similar to those reported in yeast.…”

Delineating the position ofrad4+/cut5+ within the DNA-structure checkpoint pathways inSchizosaccharomyces pombe

“…Rad9 forms a complex with Hus1 and Rad1 and is itself phosphorylated in response to DNA damage. Both of these post-translational modification events are dependent on the remaining checkpoint Rad proteins but independent of Crb2 (also termed Rhp9) and the effector kinases (Kostrub et al, 1998;Caspari et al, 2000a;Caspari et al, 2000b). Fig.…”

“…These proteins are fundamental to both pathways and appear necessary for the receipt and transmission of the checkpoint signal (Al-Khodairy and Carr, 1992;Enoch et al, 1992;O'Connell et al, 2000). As yet, the precise mechanism(s) by which these proteins act to achieve this goal is unclear, although recent studies have indicated the presence of discrete intracellular complexes between Rad3 and Rad26, between Rad17 and the four small subunits of replication factor C (RFC) and between Rad9, Rad1 and Hus1 (Edwards et al, 1999;Shimada et al, 1999;Kostrub et al, 1998;Caspari et al, 2000a). The Rad3-Rad26 complex functions as a PI3-related protein kinase and the Rad1-dependent complex (known as the 9-1-1 complex) is related to the proliferating cell nuclear antigen (PCNA) sliding clamp.…”

“…Activation and phosphorylation of Cds1 is S-phasespecific in response to either inhibition of DNA replication or DNA damage, and kinase activation correlates with phosphorylation of the protein , providing a useful marker for the S-M checkpoint. Rad9 and Rad26 are also phosphorylated in response to radiationinduced DNA damage and Hus1 is phosphorylated in response to both DNA damage and prolonged inhibition of DNA replication (Kostrub et al, 1998;Edwards et al, 1999;Caspari et al, 2000a;Caspari et al, 2000b). Ultimately, all of these phosphorylation events have been demonstrated to be Rad3-dependent and this has enabled hierarchical relationships between fission yeast checkpoint proteins to be determined.…”