Identification of a Human Homologue of theSchizosaccharomyces pombe rad17+ Checkpoint Gene

Abstract

In the fission yeast Schizosaccharomyces pombe the rad17 ؉ gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints. We have identified a human cDNA homologue of the S. pombe rad17 ؉ checkpoint gene, designated Hrad17. Hrad17 has 49% identity to the S. pombe rad17 ؉ sequence at the DNA level and 49% identity and 72% similarity at the amino acid level. Northern blot analysis indicates elevated levels of expression in testis and in cancer cell lines. Chromosomal localization by fluorescence in situ hybridization indicates that Hrad17 is located on chromosome 4q13.3-21.2. This region is subject to loss of heterozygosity in several human cancers. To begin to understand the protein-protein interactions of the human checkpoint machinery, we have used the yeast two-hybrid system to examine potential interactions between Hrad1, Hrad9, and Hrad17. We demonstrate a physical interaction between Hrad17 and Hrad1 but no interaction with Hrad9.

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.…”

“…Studies in S. cerevisiae have shown genetic and biochemical interactions among the members of the scRAD24 epistasis group (21,22), which includes S. cerevisiae homologs for HRAD1 (scRAD17), HRAD17 (scRAD24), and HRAD9 (partially homologous to scDDC1). Additionally, previous work demonstrated that hRad17 and hRad1 interacted in a two-hybrid system (16). Therefore, we addressed the possibility that hRad1 and hHus1 might interact with the checkpoint protein hRad17.…”

“…Cells were transfected with 40 g of pcDNA3 (lanes 1, 5, 9, 13), 30 g of pcDNA3-AU1 2 -hRad9 (lanes 3, 4,7,8,11,12,15,16), 5 g of pEF-BOS-⌬RI-hHus1-HA 2 (lanes 2, 4,6,8,10,12,14,16), or 5 g of pCDNA3-FLAG 2 -hRad1 expression vectors. Twenty hours after transfection, the cells were lysed, and an aliquot of each lysate was prepared for electrophoresis (Lysate).…”

“…The similarities between yeast and humans extend beyond these proteins, as human homologs of sprad1 (12)(13)(14), sprad9 (15), sprad17 (16), spchk1 (17), and sphus1 (18) genes have been identified, fueling speculation that the checkpoint pathways may be conserved. Despite these advances, the physical and functional roles of these proteins are largely unexplored, even in the well studied yeast systems.…”

ATM-dependent Phosphorylation of Human Rad9 Is Required for Ionizing Radiation-induced Checkpoint Activation

“…Mammalian counterparts of all the checkpoint rad genes except rad26 ϩ have been identified (21)(22)(23)(24)(25)(26)(27). The hRad9, hRad1, and hRad17 genes partially complement the DNA damage sensitivity of the counterpart yeast mutants (24,27,28), reinforcing the functional similarities between the human and yeast genes.…”