Downloaded from genesdev.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION tinho et al.1998).Rad1, Rad9, and Hus1 form a trimeric Genomic instability complex resembling the PCNA trimer that acts as a slid- and endoreduplication triggered ing clamp for DNA polymerases during DNA synthesis and interacts with proteins involved in cell-cycle con- by RAD17 deletion trol, DNA repair, and chromatin assembly (Kaur et al. 1 2,3 1 2001).The Rad17 protein is conserved with subunits of Xin Wang, Lee Zou, Huyong Zheng, the replication factor C (RFC), which loads the PCNA Qingyi Wei,4 Stephen J. Elledge,2,3,5 and Lei Li1,6,7 clamp onto primed DNA structure.In human cells, 1 Rad17 was found to be in a heteropentameric complex Departments of Experimental Radiation Oncology, with RFC subunits.Meanwhile, Rad17 also interacts 4Epidemiology, and 6Molecular Genetics, University of Texas 2 with the Rad1/Rad9/Hus1 trimer (Volkmer and Karnitz M.D. Anderson Cancer Center, Houston, Texas 77030; Verna 1999; Caspari et al.2000; Lindsey-Boltz et al.2001). & Mars McLean Department of Biochemistry and Molecular 3 5 Therefore, a checkpoint sliding clamp-loader model for Biology, Howard Hughes Medical Institute, and Department lesion recognition has been proposed (O’Connell et al. of Molecular and Human Genetics, Baylor College of 2000; Venclovas and Thelen 2000).Rad3-mediated Medicine, Houston, Texas 77030, USA Rad26 phosphorylation, however, has been observed in fission yeast in the absence of the clamp-clamp loader Cell cycle checkpoints are critical for genomic stability. proteins (Edwards et al.1999).Similarly in budding Rad17, a component of the checkpoint clamp loader yeast, the Atr/Atrip homolog, Mec1/Ddc2, and the complex (Rad17/Rfc2-5), is required for the response to checkpoint clamp and clamp loader complex (Rad17/ DNA damage and replication stress. To explore the role Ddc1/Mec3-Rad24/Rfc2-5 in budding yeast) could both of Rad17 in the maintenance of genomic integrity, we localize to the damage site independently (Kondo et al. established somatic conditional alleles of RAD17 in hu- 2001; Melo et al.2001).These observations suggest that man cells. We find that RAD17 is not only important for the clamp-loader complex may not be essential for dam- the Atr-mediated checkpoint but is also essential for cell age recognition of the Mec1 or Rad3 kinases.More re- viability. Cells lacking RAD17 exhibited acute chromo- cent studies in humans suggest that Rad17 is required for loading the Rad1/Rad9/Hus1 clamp onto damaged DNA somal aberrations and underwent endoreduplication at a and may act to facilitate Atr-dependent phosphorylation high rate. Therefore, RAD17 links the checkpoint to of downstream targets (Zou et al.2002).In this study, we ploidy control and is essential for the maintenance of have shown that the function of RAD17 is critical in the chromosomal stability. Atr-dependent checkpoint signal initiation and is also Supplemental material is available at http://www. important in the maintenance of genomic stability. genesdev.org. Results and Discussion Received December 6, 2002; revised version accepted February 19, 2003. Generation of RAD17 somatic conditional alleles The DNA damage and replication checkpoint is essen- To investigate the function of the clamp-loader complex, tial for the maintenance of genomic stability.Recent we performed gene targeting in human HCT116 colon studies have established a framework of the signal trans- epithelial cells and constructed a conditional RAD17- flox/− duction process in mammalian cells (Zhou and Elledge null cell line (RAD17 ), in which one allele of 2000; Rouse and Jackson 2002).Two protein kinases of RAD17 is disrupted by an in-frame insertion of the neo- the PI-3-related protein family, Atm and Atr, are key mycin coding sequence, whereas the second allele has components of the pathway.Both Atm and Atr are im- two lox sites flanking exon 5 (Fig.1A,B).The amount of flox/− ∼ portant for the maintenance of genomic stability.Atm is Rad17 in RAD17 cells exhibited 50% reduction be- primarily involved in the response to double-strand fore adenovirus-mediated Cre incision, indicating that DNA breaks (DSBs), whereas Atr responds to both DSBs the presence of lox sites flanking exon 5 in the condi- and other damage causing replication stress (Abraham tioned allele has minimum effect on RAD17 transcrip- 2001).Current evidence suggests that Atr and Atm are tion or splicing.Upon adenovirus-mediated Cre incision, signal-initiating kinases for the checkpoint kinase cas- a decline in the amount of Rad17 could be detected cade that leads to activation of downstream targets in- within 2 d and the level of Rad17 became nearly unde- cluding Brca1, Chk1, Chk2, p53, and Nbs1.How Atr or tectable by 4 d post infection, indicating a highly effi- Atm is activated by DNA damage and how other com- cient removal of the conditional exon 5 in the flox/− ponents of the checkpoint are involved in the activation RAD17 mutant cells (Fig.1C).As a control, cellular process, however, remains to be elucidated. levels of the Rad9 protein were unaffected by Ad–Cre In fission yeast, activation of the rad3-dependent infection.However, loss of Rad17 severely inhibited checkpoint requires Rad1, Rad9, Hus1, and Rad17 (Mar- damage-induced chromatin association of the Rad9 pro- tein, which may be important for the recruitment of down- stream targets (Zou et al.2002; Supplemental Fig.1). [Keywords: RAD17; DNA damage; checkpoint; endoreduplication] 7 RAD17 is essential for cell viability as assessed by clo- Corresponding author. nogenic analysis.We found that >90% of Ad–Cre-in- E-MAIL [email protected]; FAX (713) 794-5369. flox/− Article published online ahead of print.Article and publication date are fected RAD17 cells were unable to form colonies, at http://www.genesdev.org/cgi/doi/10.1101/gad.1065103. whereas RAD17flox/+ cells (with one wild-type RAD17 GENES & DEVELOPMENT 17:965–970 © 2003 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/03 $5.00; www.genesdev.org 965 Downloaded from genesdev.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press Wang et al. appeared to be normal in the absence of Rad17 (Fig.2D,E).Together, these results suggest that Atm activation is independent of the clamp–clamp- loader complex.Furthermore, we found that the Chk2 Thr 68 phos- phorylation was significantly en- hanced in response to UV irradiation, which normally induces a low level of Chk2 phosphorylation in wild-type cells.This result suggests that the de- fect in Atr signaling caused by loss of Rad17 may allow Atm to have a com- pensatory role in activating the Chk2 kinase on UV damage, as double- strand breaks resulting from DNA damage-induced stalled replication forks could provide an alternative route for activating the Atm kinase. Such a crossover in downstream sub- strate selection, observed in fission yeast (Martinho et al.1998), may offer secondary or compensatory protec- tion against genotoxic agents in case one DNA damage-sensing branch is Figure 1. Generation of a conditional RAD17 allele in human somatic cells.(A) Targeting compromised. strategy for establishing a conditional RAD17 allele.Construct 1 contains a duplicated exon 5 as well as an exon 5 disrupted in-frame with the complete coding sequence of the neomycin resistance (Neo) gene and polyadenylation sequence (black squares).Thr ee lox sites (triangles) Damage-induced mitotic delay is were positioned as indicated.Following targeting with Construct 1, exon 5 with the Neo defective in loss of RAD17 insertion was removed by selective Cre incision between the first two lox sites to generate the function mutant Conditional allele and allow recycle of the Neo marker.A second round of ta rgeting with Construct 2 generated the Inactivated allele.(B) Southern blot analysis of genomic DNA We next examined the radiation-in- isolated from the indicated cell lines after digestion with SpeI.+/+, RAD17 wild-type HCT116 duced G2/M checkpoint in cells lack- cells by the presence of wild-type alleles (8.1 kb); flox/+, cells that had been successfully +/+ flox/− targeted with Construct 1, showing a wild-type band and band 1 (9.6 kb); flox/−, cells with the ing Rad17. RAD17 , RAD17 , − Inactivated allele (band 3, 8.5 kb) and the Conditional allele (band 3, 7.3 kb).A SpeI site and ATRflox/ cells were infected with immediately downstream of the last lox site in both Construct 1 and 2 reduced the length of Ad–Cre and subsequently exposed to the conditional allele band (band 3) by 0.8 kb, compared with the wild-type band.(C) Immu- ␥ noblot analysis of RAD17flox/− cells 3 d after Ad–Cre and Ad–GFP infection, respectively.The -radiation.Entry into mitosis was loss of the Rad17 protein was nearly complete whereas Rad9 levels remained constant in both measured by flow cytometry com- cells. bined with phospho-histone H3 im- munofluorescent staining (Fig.3A,B). +/+ allele) showed no difference compared with RAD17+/+ In RAD17 cells, mitotic entry was effectively delayed cells, indicating that Rad17 is essential for sustained cell by radiation treatment.In RAD17flox/− cells, however, growth.However, the majority of RAD17flox/− cells re- the mitotic population was significantly higher, indicat- mained viable 5 d after Ad–Cre infection, allowing fur- ing a defective G2/M checkpoint resulting from loss of ther phenotypic analysis of the loss-of-function Rad17 Rad17.In addition to the erroneous entry into mitosis mutant (Supplemental Fig.2).