Human Rad9 is required for the activation of S-phase checkpoint and the maintenance of chromosomal stability.
In response to DNA damage or replication block, cells activate a battery of checkpoint signaling cascades to control cell cycle progression and elicit DNA repair in order to maintain genomic stability and integrity. Identified as a homolog of its fission yeast counterpart, human Rad9 was proposed to form a Rad9-Hus1-Rad1 protein complex to mediate checkpoint signals. However, the precise function of Rad9 in the process of checkpoint activation is not fully understood. Using the RNA interference technique, we investigated the role of Rad9 in the genotoxic stress-induced activation of S-phase checkpoint and the maintenance of chromosomal stability. We found that Rad9 knockdown reduced the phosphorylation of Rad17, Chk1 and Smc1 in response to DNA replication block and certain types of DNA damage. Immunofluorescence studies showed that the removal of Rad9 disrupted the foci formation of phosphorylated Chk1, but not ATR. Moreover, Rad9 knockdown resulted in radioresistant DNA synthesis and reduced cell viability under replication stress. Finally, removal of Rad9 by RNAi led to increased accumulation of spontaneous chromosomal aberrations. Taken together, these results suggest a critical and specific role of Rad9 in the activation of S-phase checkpoint and the maintenance of chromosome stability.
Dang
Tongyun
T
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.
Bao
Shideng
S
Wang
Xiao-Fan
XF
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002470:Cell Survival / Q000528:radiation effects; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D043171:Chromosomal Instability / Q000502:physiology* / Q000528:radiation effects; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication / Q000528:radiation effects; D005720:Gamma Rays; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034622:RNA Interference; D016196:S Phase / Q000502:physiology* / Q000528:radiation effects; D000098550:Structural Maintenance of Chromosome Protein 1
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C113575:Rad17 protein, human; D000098550:Structural Maintenance of Chromosome Protein 1; C069930:rad9 protein; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases