Retention of the human Rad9 checkpoint complex in extraction-resistant nuclear complexes after DNA damage.
Studies in yeasts and mammals have identified many genes important for DNA damage-induced checkpoint activation, including Rad9, Hus1, and Rad1; however, the functions of these gene products are unknown. In this study we show by immunolocalization that human Rad9 (hRad9) is localized exclusively in the nucleus. However, hRad9 was readily released from the nucleus into the soluble extract upon biochemical fractionation of un-irradiated cells. In contrast, DNA damage promptly converted hRad9 to an extraction-resistant form that was retained at discrete sites within the nucleus. Conversion of hRad9 to the extraction-resistant nuclear form occurred in response to diverse DNA-damaging agents and the replication inhibitor hydroxyurea but not other cytotoxic stimuli. Additionally, extraction-resistant hRad9 interacted with its binding partners, hHus1 and an inducibly phosphorylated form of hRad1. Thus, these studies demonstrate that hRad9 is a nuclear protein that becomes more firmly anchored to nuclear components after DNA damage, consistent with a proximal function in DNA damage-activated checkpoint signaling pathways.
Burtelow
M A
MA
Divisions of Radiation Oncology and Developmental Oncology Research and Departments of Immunology and Molecular Pharmacology, Mayo Foundation, Rochester, Minnesota 55905, USA.
Kaufmann
S H
SH
Karnitz
L M
LM
D000818:Animals; D018797:Cell Cycle Proteins / Q000032:analysis / Q000378:metabolism*; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins; D004720:Endonucleases / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D046911:Macromolecular Substances; D010766:Phosphorylation; D011817:Rabbits; D029702:Schizosaccharomyces pombe Proteins; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D029702:Schizosaccharomyces pombe Proteins; C106332:hus1 protein, S pombe; C069930:rad9 protein; D004720:Endonucleases; D006918:Hydroxyurea