Hrad17, a Structural Homolog of the Schizosaccharomyces Pombe RAD17 Cell Cycle Checkpoint Gene, Stimulates P53 Accumulation

Oncogene (1999) 18, 1689 ± 1699 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc hRAD17, a structural homolog of the Schizosaccharomyces pombe RAD17 cell cycle checkpoint gene, stimulates p53 accumulation Lei Li1, Carolyn A Peterson2, Gunilla Kanter-Smoler3, Ying-Fei Wei4, Louis S Ramagli2, Per Sunnerhagen3, Michael J Siciliano2 and Randy J Legerski*,2 1Department of Experimental Radiation Oncology, University of Texas, MD Anderson Cancer Center, Houston, Texas 77030, USA; and 2Department of Molecular Genetics, University of Texas, MD Anderson Cancer Center, Houston, Texas 77030, USA; 3Department of Molecular Biology, Lundberg Laboratory, Goeteborg University, PO Box 462, SE-405 30, Goeteborg, Sweden; 4Human Genome Sciences, Inc., Rockville, Maryland 20850, USA The RAD17 gene product of S. Pombe is an essential transition. Consequently, the cell cycle arrests at G1/ component of the checkpoint control pathway which S, S, and G2/M stages depending upon where in the responds to both DNA damage and disruption of cell cycle the damage is sensed, thus minimizing replication. We have identi®ed a human cDNA that proliferation of mutant cells and allowing damaged encodes a polypeptide which is structurally conserved DNA to be corrected by repair pathways and with the S. Pombe Rad17 protein. The human gene, interrupted replication to be completed (Elledge, designated hRAD17, predicts an encoded protein of 590 1996; Nurse, 1997; Paulovich et al., 1997). amino acids and a molecular weight of 69 kD. Amino DNA damage checkpoints in lower eukaryotes have acid sequence alignment revealed that hRad17 has been extensively investigated. In the budding yeast S. 28.3% and 52.5% similarity with the S. Pombe Rad17 Cerevisiae, scRAD9, scRAD17, scRAD24 and scMEC3 protein, and 21.8% identity and 45.8% similarity to the (the pre®xes here and after, h, sc, and sp refer to budding yeast cell cycle checkpoint protein, Rad 24. Homo sapiens, Saccaromyces cerevisiae, and Schizo- When introduced into the S. Pombe rad17 mutant, saccharomyces pombe, respectively) are required for hRAD17 was able to partially revert its hydroxyurea and the DNA damage-induced G1 and G2 arrest and S- ionizing radiation hypersensitivity, but not its UV phase regulation (Siede et al., 1994; Weinert et al., hypersensitivity. Permanent overexpression of the 1994; Paulovich and Hartwell, 1995; Paulovich et al., hRAD17 gene in human ®brosarcoma cells resulted in 1997); whereas, scPOL2, scRFC5, and scDBP11 are p53 activation and a signi®cant reduction of S- and G2/ required for the S-phase arrest induced by DNA M-phase cells accompanied by an accumulation of the replication-blocking agents (Araki et al., 1995; Navas G1-phase population, suggesting that hRAD17 may have et al., 1995; Sugimoto et al., 1996). Two kinases, a role in cell cycle checkpoint control. Immunostaining of scMec1/Esr1/Sad3 (Allen et al., 1994; Kato and HT-1080 cells transiently transfected with a hRAD17 Ogawa, 1994; Weinert et al., 1994; Sanchez et al., construct con®rmed the nuclear accumulation of p53, 1996) and scRad53/Spk1/Mec2/Sad1 (Stern et al., which mimics the induction caused by DNA damage. 1991; Allen et al., 1994; Weinert et al., 1994) appear Using FISH analysis, we have mapped the hRAD17 to be transducers of the DNA damage signal, resulting locus to human chromosome 5q11.2. in cell cycle arrest and transcriptional induction of repair genes (Elledge, 1996). In the ®ssion yeast S. Keywords: cell cycle checkpoint; DNA damage; p53; pombe, the `checkpoint-rad' group of genes including ®ssion yeast S. Pombe; RAD17; human homolog spRAD1, spRAD3, spRAD9, spRAD17, spRAD26, and spHUS1 is required for checkpoint arrest following both DNA lesions or replication blockage (Al- Khodairy and Carr, 1992; Al-Khodairy et al., 1994; Carr, 1995). The spChk1 and spCds1 kinases are Introduction required for survival upon DNA damage, or replication blockage with hydroxyurea (HU), respec- DNA damage checkpoints are signal transduction tively (Murakami and Okayama, 1995; Walworth and systems that link damage to DNA, or incomplete Bernards, 1996; Howard et al., 1998; Boddy et al., DNA synthesis, with the components of the cell cycle. 1998). spChk1 has been shown to act downstream of Signals originating from damaged DNA activate the `checkpoint rad' group as a signal transducer checkpoint pathways and target the mitotic apparatus (Ford et al., 1994; Walworth and Bernards, 1996; through a number of distinct mechanisms including: Furnari et al., 1997). induction of cdk inhibitors, down-regulation of cyclin In mammals, ®ve genes, ATM, p53, p21/WAF1/ expression, and inhibition of cdk phosphorylation/ CIP1, hCHK1, and 14-3-3s have been identi®ed as dephosphorylation required in normal cell cycle components of DNA damage-induced checkpoint pathways. The Atm kinase, p53, and hChk1 are signal processing and transmitting factors (Yin et al., 1992; Savitsky et al., 1995; Sanchez et al., 1997), while p21, *Correspondence: RJ Legerski and, possibly, 14-3-3s are eectors of checkpoint arrest Received 5 June 1998; revised 21 September 1998; accepted 6 October (El-Deiry et al., 1993; Harper et al., 1993; Hermeking 1998 et al., 1997). A member of the PI-3 kinase family, the Human homolog of S. pombe rad17 LLiet al 1690 ATM gene may actually function as a protein kinase Results (Hunter, 1995). It has proven to be involved in G1/S, S, and G2/M DNA damage checkpoints, and is an Identi®cation of the hRAD17 gene element upstream of p53, since cells lacking ATM show a reduced activation of p53 in response to DNA The S. pombe RAD17 gene is a structural homolog of damage (Kastan et al., 1992). Upon DNA damage, the scRAD24. Both genes appear to function at initial p53 cellular level, stability, and speci®c activity as a steps of checkpoint control, and the scRad24 protein transcription factor are up-regulated (Lu and Lane, has been implicated in damage processing (Lydall and 1993; Levine, 1997). One of the targets of p53 Weinert, 1995). Loss of spRAD17 in S. pombe transactivation is the p21/CIP1/WAF1 gene, a down- abrogates the G2 checkpoint and results in sensitivity stream eector of the G1 checkpoint. p21 binds and to DNA damage and replication blocking reagents inhibits a number of cyclin-dependent kinases, and (Griths et al., 1995). To identify a human homolog mediates the arrest at the G1/S transition. In addition, of spRad17, the full-length peptide sequence of it interacts with PCNA, thereby inhibiting DNA spRad17 was used to conduct a BLAST search of replication, and possibly eecting S phase arrest. The the human EST database at Human Geonome human Chk1 kinase is phosphorylated upon DNA Sciences, Inc. (HGS, Rockville, MD, USA). One damage, in a mechanism possibly mediated by Atm EST sequence possessed a predicted 60 amino acid (Nurse, 1997), and phosphorylates Cdc25C on Ser216, region with 29% identity and 56% similarity to resulting in a sequestration of Cdc25C phosphatase spRad17. The complete nucleotide sequence of the activity, which is necessary for the activation of Cdc2- 1.7 Kb EST insert was determined, and sequence cyclin B and mitotic entry. Thus far, signal transducing alignment analysis revealed that the amino acid factors and the downstream eectors of the damage sequence from the largest open reading frame shared checkpoint have been partially elucidated in mammals. homology with the C-terminal half of spRad17 as well However, upstream elements which are responsible for as scRad24. Therefore, a HeLa cDNA library was detecting damage and originating checkpoint signals screened, using the EST sequence as a probe, to obtain remain largely unknown. a full-length cDNA. The longest cDNA clone Structural and functional conservation of the DNA obtained, after screening 400 000 colonies, contained damage checkpoint genes have been observed among a 2.8 Kb insert. Sequence analysis indicated that this budding yeast, ®ssion yeast, and mammals (Elledge cDNA insert is 2812 bp in length, has 268 bp of 5' 1996; Paulovich et al., 1997), indicating that there is a untranslated region, an open reading frame of 2013 bp common molecular mechanism underlying checkpoint and 532 bp of 3' untranslated region. An inframe stop controls throughout eukaryotic systems. In particular, codon was found 30 bp upstream of the initiation signals derived from DNA damage are transmitted codon. The open reading frame encodes a predicted through conserved protein kinases common in all polypeptide of 671 amino acids, which shares three organisms as re¯ected by the sequence conserva- homology with both spRad17 and scRad24. When tion and functional similarity among scMec1, spRad3, aligned in a pairwise fashion, hRad17 exhibits closer Atm, and Atr. The Chk1 function and the involve- homology to spRad17, than to scRad24, with 28.3% ment of the 14-3-3 proteins were also shown to be identity and 52.7% similarity to the former protein, strictly conserved between ®ssion yeast and humans and 21.8% identity and 45.8% similarity to the latter. (Ford et al., 1994; Furnari et al., 1997; Peng et al., Both the scRad24 and spRad17 proteins are known 1997; Sanchez et al., 1997). Therefore, it is reasonable to exhibit signi®cant structural conservation with a that certain upstream components which are respon- family of proteins identi®ed as components of human sible for detecting DNA damage and producing initial RFC/activator 1, and with E. coli and bacteriophage checkpoint signals, such as scRAD9, scRAD17, T4 DNA polymerase accessory factors (O'Donnell et scRAD24 in S. Cerevisiae and spRAD1, spRAD9, al., 1993; Griths et al., 1995). The hRad17 protein and spRAD17 in S. pombe, may also exist and be also exhibits homology to these proteins (Figure 1). required in mammalian DNA damage checkpoints. In RFC is a multifactorial complex composed of ®ve fact, a human cDNA homologous to spRAD9 has subunits and functions as a loading factor for PCNA been isolated and was found partially active in during DNA polymerases d and e-mediated DNA rescuing a sprad9 mutant phenotype (Lieberman et synthesis (Pan et al., 1993).

Load more