cDNA Cloning and Gene Mapping of Human Homologs forSchizosaccharomyces pombe rad17, rad1,andhus1and Cloning of Homologs from Mouse,Caenorhabditis elegans,andDrosophila melanogaster

Dean, Frank B.; Lian, Lubing; O'Donnell, Mike

doi:10.1006/geno.1998.5587

Genomics

1998

DOI: 10.1006/geno.1998.5587

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cDNA Cloning and Gene Mapping of Human Homologs forSchizosaccharomyces pombe rad17, rad1,andhus1and Cloning of Homologs from Mouse,Caenorhabditis elegans,andDrosophila melanogaster

Frank B. Dean

Lubing Lian

Mike O'Donnell

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Cited by 45 publications

(40 citation statements)

References 85 publications

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“…7 hRad17 is another of the early-response elements in the DNA damage checkpoint pathway. 8,9 In the Rad17-RFC complex, the large subunit of RFC is replaced by hRad17. 3,5 hRad9 has several important properties.…”

mentioning

confidence: 99%

Accumulation of hRad9 protein in the nuclei of nonsmall cell lung carcinoma cells

Maniwa

Yoshimura

Bermudez

et al. 2004

Cancer

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BACKGROUNDDNA damage sensor proteins have received much attention as upstream components of the DNA damage checkpoint signaling pathway that are required for cell cycle control and the induction of apoptosis. Deficiencies in these proteins are directly linked to the accumulation of gene mutations, which can induce cellular transformation and result in malignant disease.METHODSUsing 48 sets of tumor tissue specimens and peripheral normal lung tissue specimens from 48 patients with nonsmall cell lung carcinoma (NSCLC) who underwent surgery, the authors investigated the expression of hRad9 protein, a member of the human DNA damage sensor family, using immunohistochemical and Western blot analyses.RESULTSImmunohistochemical analysis detected the accumulation of hRad9 in the nuclei of tumor cells in 16 tumor tissue specimens, (33% of tumor tissue specimens examined). Western blot analysis also revealed elevated levels of phosphorylated hRad9 protein in NSCLC cells that was accompanied by the detection of phosphorylated Chk1, a protein kinase that regulates the downstream signaling of the DNA damage checkpoint pathway. Furthermore, strong expression of hRad9 was correlated with an increase in Ki‐67 expression index in the tumor cells that were examined.CONCLUSIONSThe findings made in the current study suggest that Rad9 expression may play an important role in cell cycle control in NSCLC cells and may influence NSCLC cell phenotype. Cancer 2005. © 2004 American Cancer Society.

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confidence: 99%

Accumulation of hRad9 protein in the nuclei of nonsmall cell lung carcinoma cells

Maniwa

Yoshimura

Bermudez

et al. 2004

Cancer

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“…[1][2][3][4][5][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)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(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.…”

mentioning

confidence: 99%

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

Burtelow¹,

Roos-Mattjus²,

Rauen³

et al. 2001

Journal of Biological Chemistry

122

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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...

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“…Rad1 has homology with Ustilago maydis Rec1 (40), which is a checkpoint protein and a 3Ј-5Ј exonuclease, suggesting that Rad1 may participate in DNA metabolic events that are required for checkpoint activation. However, it is unclear whether human or S. pombe Rad1 also possesses nuclease activity (12,14,17). In addition, recombinant hRad9 has recently been shown to possess 3Ј-5Ј exonuclease activity (41).…”

mentioning

confidence: 99%

“…Many of the genes controlling checkpoint activation were identified by genetic studies in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Recently, homologs of the yeast checkpoint genes were identified in higher eukaryotes, suggesting that much of the checkpoint machinery is highly conserved (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Detailed biochemical and genetic studies in both mammals and yeasts provide a working model for checkpoint activation.…”

mentioning

confidence: 99%

Retention of the Human Rad9 Checkpoint Complex in Extraction-resistant Nuclear Complexes after DNA Damage

Burtelow¹,

Kaufmann²,

Karnitz³

2000

Journal of Biological Chemistry

102

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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, extractionresistant 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.In eukaryotes, DNA damage activates complex cellular responses that initiate DNA repair and slow or block progression through the cell cycle (reviewed in Refs. 1-6). Activation of cell cycle arrest is mediated by the checkpoint signaling machinery. Many of the genes controlling checkpoint activation were identified by genetic studies in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Recently, homologs of the yeast checkpoint genes were identified in higher eukaryotes, suggesting that much of the checkpoint machinery is highly conserved (7-23). Detailed biochemical and genetic studies in both mammals and yeasts provide a working model for checkpoint activation. Central to this model are the phosphatidylinositol 3-kinase-related kinases (PIKKs) 1 (1,3,4,24,25). Human cells lacking the PIKK ataxia telangiectasia mutated, the product of the ATM gene, have global checkpoint defects and are extremely sensitive to ionizing radiation (reviewed in Ref. 26). Likewise, S. pombe lacking the PIKK spRad3 and S. cerevisiae lacking scMec1 have similar phenotypes; they cannot block cell cycle progression after DNA damage and are exquisitely sensitive to diverse genotoxic agents (27,28). The PIKKs function as signal transducers that relay activating signals to downstream effector protein kinases, including spChk1 and spCds1 in S. pombe (18) and hChk1 and hChk2 (hCds1) in humans (20,22,23). Although hChk1 and hChk2 have different functions, one common downstream target is the cell-cycle phosphatase Cdc25 (22,23,29,30). Phosphorylation of Cdc25 inhibits its activity (21, 31), and phosphorylated Cdc25 is sequestered in the cytoplasm (32, 33), preventing it from activating cyclin B/Cdc2. Thus, the PIKKs function as signal transducers that relay activating signals to effector protein kinases, which then block the G 2 /M transition.The PIKKs are not the only components of the DNA damageactivated signaling pat...

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cDNA Cloning and Gene Mapping of Human Homologs forSchizosaccharomyces pombe rad17, rad1,andhus1and Cloning of Homologs from Mouse,Caenorhabditis elegans,andDrosophila melanogaster

Cited by 45 publications

References 85 publications

Accumulation of hRad9 protein in the nuclei of nonsmall cell lung carcinoma cells

Accumulation of hRad9 protein in the nuclei of nonsmall cell lung carcinoma cells

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

Retention of the Human Rad9 Checkpoint Complex in Extraction-resistant Nuclear Complexes after DNA Damage

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