Oncogene (2000) 19, 5291 ± 5297 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc SHORT REPORT PCNA interacts with hHus1/hRad9 in response to DNA damage and replication inhibition

Kiyoshi Komatsu1, Walker Wharton2, Haiying Hang3, Chun Wu4, Sujay Singh4, Howard B Lieberman3, WJ Pledger2 and Hong-Gang Wang*,1

1Drug Discovery Program, H. Lee Mott Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, Florida, FL 33612, USA; 2Molecular Oncology Program, H. Lee Mott Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, Florida, FL 33612, USA; 3Center for Radiological Research, Columbia University, 630 West 168th Street, New York, NY 10032, USA; 4Imgenex Corporation, 11175 Flintkote Avenue, San Diego, California, CA 92121, USA

The hHus1 and several hRad proteins are involved in the et al., 1998), suggesting that the DNA integrity control of DNA integrity checkpoints, although the checkpoint pathway controlled by these proteins may mechanisms underlying these processes are unknown. be conserved from yeast to human. Many of these Using a yeast two-hybrid system to detect protein- proteins can interact with each other through a protein interactions, we found that human proliferating complex network of homo- and heterodimers (Hang cell nuclear antigen (PCNA), a protein known to and Lieberman, 2000; Kondo et al., 1999; Kostrub et function in both DNA replication and repair, interacts al., 1997; St. Onge et al., 1999; Volkmer and Karnitz with the human checkpoint-related protein Hus1 1999). (hHus1). In human skin ®broblast cells, exposure to The S. pombe hus1 gene was originally isolated from ionizing radiation of hydroxyurea triggers translocation a screen for ®ssion yeast mutants capable of entering of hHus1 from the cytosol to the nucleus, where it mitosis when DNA replication is blocked by hydro- associates with PCNA as well as another checkpoint xyurea (HU) (Enoch et al., 1992). However, the hus1 protein, hRad9. This nuclear translocation and the mutants are not only abnormally sensitive to HU but complex formation or hHus1 with PCNA and hRad9 also sensitive to radiation exposure, suggesting that correlate closely with changes in cell cycle distribution in many of these genes required for cell-cycle arrest in response to radiation exposure. These results suggest response to blocked DNA replication are also required that this multi-protein complex may be important for for DNA damage checkpoint control. The Hus1 coordinating cell-cycle progression, DNA replication and protein interacts with Rad1 and Rad9 (Caspari et al., repair of damaged DNA. Oncogene (2000) 19, 5291 ± 2000; Kondo et al., 1999; Kostrub et al., 1997), 5297. forming a large protein complex *450 kDa that is much bigger than an expected trimeric size of Keywords: hRad9; hHus1; PCNA; DNA damage; cell- *150 kDa, implying a possibility that this multi- cycle checkpoints protein complex might contain other unidenti®ed members. Similar interactions have been reported for hHus1, hRad1 and hRad9, strongly suggesting that these human homologs are functionally equivalent to Cell-cycle checkpoints play a critical role in the their S. pombe counterparts. Interestingly, the reported maintenance of genomic integrity by inhibiting pro- ®ndings that Hus1, Rad1 and Rad9 as well as their gression through the cell cycle in the presence of human homologs share regions of sequence similarity damaged DNA or incomplete DNA replication with the proliferating cell nuclear antigen (PCNA) (Hartwell and Kastan, 1994; Russell, 1998). The loss (Aravind et al., 1999; Caspari et al., 2000; Thelen et al., of genomic integrity can cause cancer and other genetic 1999; Venclovas and Thelen, 2000) suggest that these diseases (Hartwell and Kastan, 1994; Nojima, 1997). In checkpoint proteins may directly impinge on the DNA ®ssion yeast, a group of six checkpoint proteins, replication machinery. The biochemical mechanism by including Hus1, Rad1, Rad3, Rad9, Rad17, and which these proteins regulate the DNA integrity Rad26, are required to block entry into mitosis when checkpoint pathways, however, remains essentially DNA replication is inhibited or DNA is damaged (al- unknown. Khodairy and Carr, 1992; al-Khodairy et al., 1994; To identify cDNAs encoding proteins that are Enoch et al., 1992; Rowley et al., 1992). Human capable of interacting with human Hus1 (hHus1), we homologs of all of these key checkpoint regulators, used a lexA-based yeast two-hybrid system (Golemis et with the exception of Rad26, have been identi®ed (i.e., al., 1994) for Matchmaker cDNA library screening. hHus1, hRad1, ATR, hRad9, and hRad17; Cimprich Strain EGY48 S. cerevisiae cells were transformed with et al., 1996; Hang and Lieberman, 2000; Kostrub et al., plasmid pEG202 containing a full-length hHus1 1998; Lieberman et al., 1996; Parker et al., 1998; Udell subcloned in frame with the LexA open reading frame (ORF) and human cDNA libraries (fetal brain and Hela mixture) cloned into pJG4-5 (Clontech Lab, Inc). *Correspondence: H-G Wang Candidate clones were isolated from yeast colonies Received 12 June 2000; revised 24 August 2000; accepted 30 formed on leucine-de®cient agar plates with detectable August 2000 b-galactosidase activity and retested by co-transforma- Interaction of PCNA with hHus1 and hRad9 K Komatsu et al 5292

Figure 1 Speci®c interaction of hHus1 with PCNA. (a) S. cerevisiae EGY48 cells were transformed with various combinations of the pEG202 expression plasmids producing LexA DNA-binding domain (BD) fusion proteins and pJG4-5 plasmids encoding B42 transactivation domain (AD) fusion proteins (Estojak et al., 1995; Komatsu et al., 2000). Two-hybrid interactions between these fusion proteins were determined by using lacZ reporter genes under the control of lexA operators. Three independent transformants were tested for their ability to produce blue color on X-Gal plates containing either galactose (Gal) or glucose (Glu), to either induce or repress, respectively, the gal-1 promoters in the two-hybrid plasmids. The interaction between LexA-BAD and AD-Bcl-xL served as a positive control in the yeast two-hybrid assays (Komatsu et al., 2000). (b, c) Exponentially growing 293T cells in 10 ml of DMEM containing 5% FCS were transiently co-transfected with 5 mg pFlag-CMV2 encoding human PCNA and 5 mg pcDNA3- HA-hHus1 expression plasmid. Cells were lysed after 2 days in 0.65 ml of ice-cold lysis bu€er (50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM NaF, 30 mM Na4P2O7,1mM EDTA, 1% Triton X-100) containing protease and phosphatase inhibitors (Komatsu et al., 2000) and immunoprecipitations were performed by incubating 0.2 ml of lysate with 20 ml of protein G-Sepharose preadsorbed with 5 mg of the indicated monoclonal antibodies. The resulting immune complexes were washed three times in the same bu€er, and subjected to 12% SDS ± PAGE/immunoblot analysis using antibodies speci®c for PCNA (Santa Cruz Biotech, Inc.) or hHus1 and detected by an enhanced chemiluminescence (ECL) system (Amersham). The anti-hHus1 polyclonal rabbit antiserum was generated by using a synthetic peptide corresponding to amino acids 215 to 233 of the hHus1 protein (NH2-ser-glu-ser-thr-his-glu-asp-arg-asn- val-glu-his-met-ala-glu-val-his-ile-asp-COOH). (d) 293T cells were transiently transfected with 10 mg pcDNA3-HA-hHus1 and immunoprecipitation was performed using monoclonal antibodies speci®c for PCNA, Bcl-xL or HA, followed by immunoblot

Oncogene Interaction of PCNA with hHus1 and hRad9 K Komatsu et al 5293 tion with the bait expressing plasmids. In a screen of homologs do not mediate or bridge the interaction of *16107 independent transformants, we isolated a hHus1 with PCNA. The association between PCNA partial cDNA encoding the C-terminal portion (amino and hHus1 was speci®c as judged by the failure of acids 83 ± 261) of human PCNA. PCNA is a ring- these proteins to form two-hybrid interactions with the shaped protein that encircles DNA and functions as a parental `empty' vectors or unrelated proteins (not DNA sliding clamp for the replicative DNA poly- shown). merases (Kong et al., 1992; Krishna et al., 1994). It is Next, we determined whether the interaction of an essential component for eukaryotic chromosomal PCNA and hHus1 could occur in mammalian cells. DNA replication machinery and plays a critical role in 293T cells were transiently co-transfected with expres- DNA recombination, repair, and several other cellular sion plasmids encoding Flag-tagged PCNA and processes (Kelman and Hurwitz, 1998; Stillman, 1994; hemagglutinin (HA)-tagged hHus1, and immunopreci- Tsurimoto, 1999). The human PCNA ORF was pitates were prepared using anti-Flag and anti-HA ampli®ed by the polymerase chain reaction (PCR) monoclonal antibodies, or with anti-Myc antibody as from a HepG2 cDNA library, and introduced into a negative control, and subjected to SDS ± PAGE/ yeast cells to characterize further the interaction of immunoblot assays using anti-sera speci®c for PCNA PCNA and hHus1. As shown in Figure 1a, PCNA, or hHus1. Under these conditions, PCNA was co- whether fused to a LexA DNA-binding domain (LexA- immunoprecipitated with hHus1 (Figure 1b,c). In PCNA) or a B42 transactivation domain (AD-PCNA), addition, endogenous PCNA could be co-immunopre- interacted with hHus1, as determined by a b- cipitated with HA-tagged or Flag-tagged hHus1 from galactosidase activity assay. In contrast, LexA-PCNA 293T cells expressing HA-hHus1 (Figure 1d) or Flag- protein failed to form two-hybrid interactions with hHus1 (Figure 1e), but not with the unrelated protein AD-hRad1 and AD-hRad9, suggesting that their yeast Bcl-xL (Figure 1d). The speci®city of the association

Figure 2 The hHus1 protein localizes in the cytosol of 293 cells. 293 cells were transiently transfected with pEGFP-hHus1 (b), pFlag-hHus1 (d), parental pEGFP-C2 (a) and pFlag-CMV2 (c) plasmid DNA, or left untransfected (e,f). In (c ± f), cells were ®xed in methanol at 7208C for 10 min and permeabilized in 0.5% Triton X-100 at room temperature for 10 min. After preblocking in 5% (v/v) skim milk and 3% (v/v) BSA for 1 h, cells were incubated with anti-Flag M2 mouse monoclonal antibody (c and d;50mg/ml, Sigma), anti-Hus1 rabbit polyclonal antiserum (f; 1 : 100) or normal rabbit serum (e), followed by rhodamine-conjugated goat anti- mouse IgG (c and d; 1 : 100, Chemicon) or ¯uorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (e and f), and analysed using ¯uorescence microscopy

analysis of the resulting immune complexes using anti-PCNA polyclonal antibody as a probe. (e) 293T cells were transiently transfected with 10 mg pFlag-hHus1 or parental pFlag-CMV2(CNTL) plasmid DNA. Immunoprecipitation was performed 2 days later using anti-Flag M2 monoclonal antibody, followed by immunoblot analysis of the resulting immune complexes with anti- PCNA polyclonal antiserum

Oncogene Interaction of PCNA with hHus1 and hRad9 K Komatsu et al 5294 between hHus1 and PCNA was further shown by the To investigate the cellular localization of hHus1, we failure of the anti-Flag (Figure 1e) or anti-HA (not expressed hHus1 as a green ¯uorescent protein (GFP) shown) antibodies to coimmunoprecipitate with fusion protein in 293 epithelial cells. Analysis of 293 PCNA in 293T cells transfected with control empty cells by ¯uorescence microscopy after transfection with vector. an expression plasmid encoding the GFP-hHus1

Figure 3 DNA damage or replication inhibition induced hHus1 translocation into the nucleus and binding to PCNA. (a ± i) Logarithmically growing Flow2000 cells were cultured without (a ± c) or with 1 mM HU for 12 h (d ± f), or exposed to 8 Gy IR followed by 5 h culture (g to i). Cells were ®xed and stained with monoclonal mouse anti-PCNA (1 : 20; Santa Cruz Biotech, Incorporated) and polyclonal rabbit anti-hHus1 (1 : 100), followed by rhodamine-conjugated goat anti-mouse IgG and FITC- conjugated goat anti-rabbit IgG (1 : 100; Chemicon), and analysed using a confocal laser-scanning microscope (Carl Zeiss). Control immunoglobulin G1 was also used for con®rming the speci®city of immunostaining (not shown). (j) Flow2000 cells were cultured for 12 h with or without 1 mM HU, as indicated (+) or (7), respectively. Cell lysates were normalized for protein content and immunoprecipitations were performed using anti-hHus1 antiserum or normal rabbit antiserum (CNTL) as a negative control. The resulting immune complexes were subjected to SDS ± PAGE and immunoblot analysis with speci®c antibodies for PCNA or hHus1. In addition to immune complexes, the total lysates (50 mg) were analysed directly, con®rming no signi®cant changes in PCNA and hHus1 expression after HU treatment. (k) Logarithmically growing Flow2000 cells were exposed to 8 Gy IR or left untreated (7). At various times after ionizing radiation exposure, cell lysates were made and anti-hHus1 immunoprecipitates were prepared, followed by immunoblot analysis using antibodies speci®c for PCNA or hHus1. The cell lysates (50 mg) were also applied directly to the gel without immunoprecipitation, con®rming that ionizing radiation had no e€ect on protein expression levels of PCNA and hHus1

Oncogene Interaction of PCNA with hHus1 and hRad9 K Komatsu et al 5295 protein revealed a di€use, mostly cytosolic staining radiation. We used Flow2000 cells to monitor changes (Figure 2b). In contrast, when the GFP protein was in the intracellular locations of endogenous PCNA and expressed without the appended hHus1 cDNA insert, a hHus1 proteins in response to DNA damage and mostly di€use pattern of ¯uorescence was found inhibition of DNA synthesis. Immuno¯uorescence throughout the cell (Figure 2a). Similar results were analysis of methanol-®xed exponentially growing cells obtained when using the Flag-tagged hHus1 protein revealed a punctate nuclear immunostaining of PCNA (Figure 2d). The mostly cytosolic distribution of hHus1 protein (Figure 3a), consistent with a localization in 293 cells was further shown by immuno¯uorescence con®ned to the sites of DNA replication (Bravo and staining of the endogenous hHus1 protein with anti- Macdonald-Bravo, 1987). Cells labeled with anti- hHus1 speci®c antibody (Figure 2f). The staining of hHus1 antibody showed that hHus1 was located control vector-transfected cells (Figure 2c) or using throughout the cell (Figure 3b), and in general failed normal rabbit serum (Figure 2e) con®rmed the to co-localize with PCNA (Figure 3c). In contrast, speci®city of these results. In contrast, the murine exposure of Flow2000 cells to the DNA synthesis Hus1 protein was detected mostly in the nucleus of inhibitor hydroxyurea (HU) or IR resulted in re- Balb/c 3T3 cells using the same rabbit antiserum (not localization of hHus1 to the nucleus and co-localiza- shown). Moreover, the S. pombe Hus1 protein has been tion with PCNA (Figure 3e,f,h,i). These results were reported to be a nuclear protein, but this nuclear further con®rmed by co-immunoprecipitation experi- localization of Hus1 requires the presence of Rad9 and ments, in which the protein complex of endogenous Rad17 (Caspari et al., 2000). These results indicate that hHus1 and endogenous PCNA could be detected when the cellular localization of hHus1 may di€er from those DNA was damaged or the replication of DNA was found in murine and yeast cells, but we do not exclude blocked (Figure 3j,k). Nuclear translocation of hHus1 the possibility that it is also mammalian cell type and association with PCNA were evident within 4 h dependent. and persisted for at least 12 h after IR exposure Flow2000 cells are a precursor diploid strain of (Figure 3h,i,k). However, a constitutive interaction human skin ®broblasts that undergoes rapid cell cycle between hHus1 and PCNA was observed when both arrest in the absence of apoptosis following exposure to proteins were overexpressed (Figure 1), thus suggesting ionizing radiation (IR), thus providing a model that enforced expression may bypass the in vivo control commonly used to investigate the cellular response to of endogenous hHus1 and PCNA shown in Figure 3.

Figure 4 hRad9 co-localizes with PCNA and hHus1. (a ± c) Logarithmically growing Flow2000 cells were ®xed and double-stained with mouse anti-hRad9 monoclonal antibody (1 : 50; Imgenex) and rabbit anti-PCNA polyclonal antiserum (1 : 100; Santa Cruz Biotech, Inc.), followed by rhodamine-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG secondary antibodies. (d ± l) Flow2000 cells were cultured as described in Figure 3 and stained with antibodies speci®c for hRad9 (d, g and j)or hHus1 (e, h and k), followed by application of either rhodamine- or FITC-conjugated secondary antibodies, to detect hRad9 or hHus1, respectively

Oncogene Interaction of PCNA with hHus1 and hRad9 K Komatsu et al 5296 It has been shown that hHus1 can interact with Table 1 E€ects of ionizing radiation on the cell cycle distribution of hRad9 and hRad1, forming a DNA damage responsive Flow2000 cells protein complex (Hang and Lieberman, 2000; St. Onge Time (h) G0/G1 S G2/M et al., 1999; Volkmer and Karnitz, 1999). We explored 0473218 the possibility that hRad9 might reside in the same 3414216 complex formed by PCNA and hHus1 in the presence 6275320 of incomplete DNA replication or DNA damage. 9283838 Consistent with previous reports (Komatsu et al., 12 29 8 62 2000; St. Onge et al., 1999), hRad9 protein was Cultures of logarithmically growing Flow2000 cells were exposed to detected mostly in the nucleus, and co-localized with 8 Gy of ionizing radiation. At various times, cells were ®xed, PCNA in Flow2000 cells as measured by indirect permeabilized, and treated with RNase before incubation in 50 mg/ml immuno¯uorescence microscopy (Figure 4a,c). In propidium iodide and DNA content analysis by ¯ow cytometry as contrast, the co-localization of hRad9 and hHus1 was described previously (Komatsu et al., 2000) barely detected in logarithmically growing Flow2000 cells (Figure 4f), although association was detected when cells were exposed to HU or IR (Figure 4i,l), 1998). In ®ssion yeast, Rad17 forms a protein complex implying that hHus1 forms a complex with PCNA and that is distinct from the Rad9/Hus1/Rad1 complex, but hRad9 when DNA replication is inhibited or DNA is it is necessary to locate this protein complex in the damaged. We have previously reported that treating nucleus (Caspari et al., 2000). Thus, it will be Hela cells with methyl methanesulphonate (MMS) interesting to determine whether hRad17 functions as resulted in perinuclear localization of Flag-hRad9, co- a molecular matchmaker to generate the linkage localization with GFP-Bcl-2, and apoptosis induction between the hRad9/hHus1/PCNA complex and DNA (Komatsu et al., 2000). In Flow2000 cells, IR failed to in replication forks or sites of DNA repair. induce re-localization of endogenous hRad9 (Figure 4j) PCNA can interact with a large number of proteins, and subsequent apoptosis (not shown). It is possible including DNA polymerases d and e, RFC, FEN1 that hRad9 controls several downstream pathways: nuclease, DNA ligase I, MLH1/MSH2, p21WAF1, p57, when damaged DNA is potentially repairable, hRad9 cyclin D, and the damage induced protein Gadd45 forms a nuclear protein complex with hHus1, hRad1, (Kelman and Hurwitz, 1998; Stillman, 1994; Tsurimo- and possibly PCNA to arrest the cell cycle, providing to, 1999), suggesting that it is an adapter molecule extra time for repair before cells re-enter the cell cycle capable of linking cellular factors to their DNA and progress through critical phases; when cells are substrates for DNA replication and repair, cell-cycle severely damaged, hRad9 could migrate out of the progression or cellular di€erentiation. It has been nucleus to trigger apoptosis through binding to Bcl-2. reported that mutations in PCNA increased the Activation of the DNA-damage checkpoint transi- sensitivity of yeast cells to DNA damage (Ayyagari et ently delays cell cycle progression by slowing move- al., 1995; Eissenberg et al., 1997), indicating involve- ment through S phase and arresting cells in the G1 and ment of PCNA in cellular DNA replication/repair. In G2/M phases. We analysed propidium-iodide-stained addition, PCNA has been shown to participate in a logarithmically growing Flow2000 cells by ¯ow replication checkpoint that controls entry into mitosis cytometry to determine the alterations in the distribu- (Waseem et al., 1992), suggesting that the formation of tion of DNA content after ionizing radiation. As the PCNA/hHus1/hRad9 complex might mediate this expected, DNA damage triggered cells to begin to checkpoint. Although the precise roles of this complex accumulate in S phase of the cell cycle at 3 h post- have to be elucidated, it may contribute to coordina- irradiation, reaching a maximum at 6 h, and slowly tion of cell cycle progression and DNA replication by passing through S phase, then arresting in G0/G1 and decreasing the eciency of replication fork movement, G2/M phase 12 h after ionizing radiation exposure providing extra time for DNA repair or recruitment of (Table 1). This correlates closely with the nuclear replication factors. translocation of hHus1 and its interaction with PCNA and hRad9 in response to DNA damage (Figures 3 and 4), suggesting that the PCNA/hHus1/hRad9 complex may contribute to coordination of cell cycle progres- Acknowledgements sion and DNA replication. We thank Nikola Valkov for assistance with ¯uorescence Rad17 has been reported to contain domains of confocal microscopy, the Molecular Biology, Flow Cyto- sequence similarity with the replication factor C (RFC) metry and Molecular Imaging core facilities at the Mott (Griths et al., 1995), a replication fork associated Cancer Center and Research Institute, and the NIH protein that is required for the loading and unloading (CA82197, CA72694, GM52493, and CA68446) for sup- of PCNA onto and o€ the DNA (Waga and Stillman, port.

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