Tipin and Timeless form a mutually protective complex required for genotoxic stress resistance and checkpoint function Danny M. Chou and Stephen J. Elledge* Department of Genetics, Howard Hughes Medical Institute, Center for Genetics and Genomics, Brigham and Women’s Hospital, Harvard University Medical School, Boston, MA 02115 Contributed by Stephen J. Elledge, October 20, 2006 (sent for review October 11, 2006)

DNA damage 兩 S phase 兩 cell cycle control 兩 circadian rhythms

T

imeless is a protein originally identified in Drosophila that controls the circadian rhythms of flies (1). Orthologs have been found in several species including mammals and Caenorhabditis elegans (2–4). A structurally related paralog in Drosophila, Timeout, has been identified (5). This is an essential gene in mice (6). In C. elegans the Timeless protein, Tim1, may actually be more functionally related to Timeout and has been shown to play an important role in sister chromatid cohesion (4). A key insight into the function of Timeless and Timeout came through the identification of homologs in yeast. The budding yeast homolog is Tof1 (7, 8), and the fission yeast homolog is Swi1 (9–12). Tof1 has been found to work in the Mrc1 (Claspin) pathway, which responds to replication stress (7, 13, 14). In the absence of Tof1 or Mrc1, replication stress creates DNA damage that is funneled into the Rad9 pathway, which normally does not respond to DNA replication stress (7, 13). Mrc1 or Tof1 mutants treated with hydroxyurea (HU) delay activation of Rad53, and this delayed activation is Rad9-dependent (7, 13). Swi1 mutants in fission yeast compromise Cds1 activation after HU treatment (12). In addition, Tof1 is required in budding yeast for the pausing of replication forks at replication fork barriers, whereas Swi1 prevents replication fork collapse (12, 14–16). Tipin is a 301-aa protein originally identified in a yeast two-hybrid screen for proteins that interact with Timeless, and ectopically expressed mouse Tipin and Timeless proteins coimmunoprecipitate (17). It remains unclear, however, what function Tipin plays in mammalian cells. The homologs of Tipin have been identified as Csm3 in budding yeast and Swi3 in fission yeast (9, 18). Like their mammalian counterpart, Csm3 and Swi3 have been shown to interact with Tof1 and Swi1, respectively, and are also required for activation of Rad53 and Cds1 in response to HU (19–24). Like Tof1, Csm3 was found to be required for the pausing of replication forks at replication fork barriers and site-specific replication termini in yeast (15, 25). Recently, human Timeless has been reported to play a role in the DNA damage checkpoint response (26). Together, the function of Tipin homologs in yeast suggests that Tipin, too, may mediate replication fork pausing in human cells, perhaps in response to replication stress, and may participate in the DNA

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damage checkpoint response. Here we explore the role of Tipin in DNA damage responses. Results Expression and Localization of Tipin. To investigate the function of

Tipin in human cells, we raised an antibody against the full-length Tipin purified from bacteria. Although Tipin has a molecular mass of 34 kDa, the affinity-purified antibody to Tipin unexpectedly recognized a specific band near 48 kDa (Fig. 1A). This band disappears upon treatment with siRNAs targeting Tipin, confirming the specificity of this antibody (Fig. 1B). Transfection with an expression construct that expresses Tipin fused to a 14.5-kDa epitope tag produces an additional band at ⬇63 kDa, further demonstrating that Tipin migrates slower than expected on SDS/ PAGE gels. To examine the expression of Tipin in the cell cycle, HeLa cells were released from a thymidine block into nocodazole, harvested at the indicated time points after nocodazole release, and subjected to Western blot analysis. As shown in Fig. 1C, Tipin levels remained constant throughout the cell cycle but exhibited a mobility shift in mitotic cells. The identity of this modification is unknown, but, in mitotic cells, Tipin is excluded from the chromatin (see below). It has been shown that the mTipin–myc fusion protein, when transiently overexpressed in NIH 3T3 cells by transfection, was detected in both nuclear and cytoplasmic compartments of the cell by immunofluorescence (17). To determine the localization of endogenous Tipin, we stained asynchronous HeLa cells with both the affinity-purified Tipin antibody and DAPI. Tipin exhibited a strictly nuclear staining throughout the cell cycle that colocalized with chromatin and was excluded from the nucleolus (Fig. 1D). The punctate staining suggests that Tipin is likely to be spatially organized within the nucleus through association with uncharacterized factor(s), and not simply to be randomly distributed within the compartment. Interestingly, upon mitotic entry, Tipin becomes dispersed evenly throughout the intracellular space. Because it is thought to be associated with chromatin during the S phase, this dispersion might be regulated, possibly by the mitosis-specific modification of Tipin. To determine the effect of Tipin depletion, we transfected either HeLa or U2OS cells with Tipin siRNA and analyzed cell cycle profiles by flow cytometry. HeLa cells transfected with Tipin siRNA exhibited a reproducible increase in S-phase cells, Author contributions: D.M.C. and S.J.E. designed research; D.M.C. performed research; D.M.C. and S.J.E. analyzed data; and D.M.C. and S.J.E. wrote the paper. The authors declare no conflict of interest. Freely available online through the PNAS open access option. Abbreviations: HU, hydroxyurea; IR, ionizing radiation; DSB, double-strand break. *To whom correspondence should be sent at: Department of Genetics, Center for Genetics and Genomics, Howard Hughes Medical Institute, Room 158D, New Research Building, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail: [email protected]. © 2006 by The National Academy of Sciences of the USA

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Tipin is a mammalian protein that interacts with Timeless, which plays a role in DNA damage checkpoint responses. Here, we show that Tipin is a nuclear protein that associates with the replicative helicase and protects cells against genotoxic agents. Tipin is required for efficient cell cycle arrest in response to DNA damage, and depletion of Tipin renders cells sensitive to ionizing radiation as well as replication stress. Loss of Tipin results in spontaneous ␥-H2AX foci, a marker for DNA double-strand breaks. We find that Tipin and Timeless form a complex that maintains the level of both proteins in cells and that the loss of either one will lead to the loss of the interacting partner. This observation explains the similar checkpoint phenotypes observed in both Tipin- and Timeless-depleted cells.

Fig. 1. Expression and localization of Tipin in proliferating cells. (A) Lysates from HeLa cells transfected with either an empty vector or a vector that expresses epitope-tagged Tipin were analyzed by Western blotting using the anti-Tipin antibody. (B) Lysates from HeLa cells transfected with control or Tipin siRNAs were analyzed by Western blotting. (C) HeLa cells were synchronized by sequential thymidine and nocodazole blocks, harvested at the indicated time points after nocodazole release, and analyzed by Western blotting using the indicated antibodies. (D) HeLa cells were grown overnight on coverslips and costained with the anti-Tipin antibody (green) and DAPI (blue). (E) HeLa or U2OS cells treated with either control or Tipin siRNAs were stained with propidium iodide (for DNA content) and analyzed by flow cytometry. Error bars indicate the standard deviation from three independent samples.

whereas U2OS cells reproducibly increased the number of G2 cells in the population (Fig. 1E). These results likely reflect differences between the two cell lines in their ability to respond to endogenous stress (see below). Endogenous Tipin Interacts with both Timeless and Mcm Proteins. The yeast orthologs Tof1 and Csm3 have been shown to interact (19, 21, 22). In mammalian cells, coimmunoprecipitation studies have also shown that ectopically expressed epitope-tagged constructs of mTipin and mTimeless can interact (17). To determine whether endogenous Tipin and Timeless interact, lysates were collected from HEK 293T cells and subjected to immunoprecipitation with either a control or anti-Tipin antibodies. As expected, endogenous Tipin was immunoprecipitated by antiTipin but not control antibodies (Fig. 2). Importantly, endogenous Timeless was found to coimmunoprecipitate specifically with Tipin in cells, in the presence or absence of HU, ultraviolet (UV) radiation, or ionizing radiation (IR) (Fig. 2 and data not shown). Furthermore, Mcm6 and Mcm7, members of the putative replicative helicase, also associated specifically with Tipin (Fig. 2). These results strongly suggest that Tipin associates not only with Timeless, but also with the replication machinery in human cells. This confirms studies in yeast that demonstrated an interaction between Tof1/Csm3 and members of the replication machinery and demonstrates that these relationships are conserved through evolution (14, 22, 27). Furthermore, these data are consistent with the possibility that Tipin, like its counterparts in yeast, plays a role in mediating replication fork pausing during the S phase. Tipin Reduction Leads to Increased Sensitivity to both DNA Damage and Replication Stress. To explore a role for Tipin in responding to

DNA damage and replication stress, siRNA-treated cells were 18144 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0609251103

assayed for sensitivity to either IR exposure or replication stress induced by HU. HeLa cells were transfected twice within a 24-h period with a control siRNA, Tipin siRNA, or ATM siRNA, were plated at low density, and were either mock-treated or exposed to various doses of IR. The surviving colonies were stained and counted 2 weeks later. As shown in Fig. 3A, survival of ATM siRNA-treated cells was significantly compromised after exposure to either 4- or 10-Gy IR compared with control siRNA-treated cells. To a lesser degree, Tipin reduction also rendered the cells sensitive to IR, particularly at the higher dose of IR. Similar results were

Fig. 2. Endogenous Tipin associates with Timeless and Mcm proteins. Equal amounts of cell lysates from mock-treated or HU-treated (10 mM for 1 h) cells were immunoprecipitated with either anti-Tipin or anti-GAPDH (as control) antibodies. To rule out coprecipitation of proteins linked only by DNA, lysates were treated with Benzonase to digest cellular DNA. Precipitated proteins were immunoblotted with the indicated antibodies to detect coimmunoprecipitating proteins.

Chou and Elledge

Fig. 3. Tipin deficiency sensitizes cells to DNA damage and replication stress and compromises both the G2/M and intra-S-phase DNA damage checkpoints. (A) HeLa cells were transfected with control, Tipin, or ATM siRNAs, plated at low density, and treated with the indicated doses of IR. Colonies were counted 2 weeks later. (B) HeLa cells were transfected with control, Tipin, or Timeless siRNAs, plated at low density, and treated with 2 mM HU for 16 h. Colonies were counted 2 weeks later. (C) U2OS cells were transfected with the indicated siRNAs and either mock-treated or exposed to 3 Gy of ␥-irradiation 1 h before harvesting. Mitotic cells were detected by propidium iodide and phosphohistone H3 staining and analyzed by flow cytometry. Percentages of mitotic cells and their levels normalized to control (in parentheses) are shown. (D) Control, ATR, Tipin, or Timeless siRNA-treated U2OS cells were exposed to the indicated levels of ␥-irradiation 1 h before harvesting. Mitotic cells were detected by propidium iodide and phosphohistone H3 staining and analyzed by flow cytometry. (E) U2OS cells transfected with control, ATR, Tipin, or Timeless siRNAs were exposed to 10 Gy of ␥-irradiation and assayed for DNA synthesis 30 min later by [3H]thymidine incorporation. The amount of DNA synthesis after irradiation is expressed as a percentage of the level in untreated cells. Error bars indicate standard deviation from three independent experiments.

Tipin Is Required for G2/M and Intra-S-Phase DNA Damage Checkpoints. To determine whether DNA damage signaling is com-

promised upon Tipin depletion, we examined the G2/M checkpoint in Tipin siRNA-treated cells. U2OS cells were transfected with the indicated siRNA twice within a 24-h period and were either mock-treated or subjected to 3-Gy IR ⬇48 h after the second transfection. Cells were harvested 1 h after treatment and analyzed by flow cytometry for histone H3 phosphorylation as a marker for entry into mitosis. As expected, very few of the control siRNA-treated cells entered mitosis after 3-Gy IR (Fig. 3 C and D). In contrast, a significant portion of Tipin siRNAtreated cells and Timeless siRNA-treated cells entered mitosis after 3-Gy IR compared with the untreated controls (Fig. 3 C and D). Even at the higher dose of 10 Gy, the G2/M DNA damage checkpoint defect was clearly apparent in Tipin- or Timelessdepleted cells (Fig. 3D). Our results therefore indicate that depletion of Tipin and Timeless significantly compromises the G2/M DNA damage checkpoint. The interaction between Tipin and Mcm proteins, as well as sensitivity to HU-induced replication stress after siRNA depletion of Tipin, led us to hypothesize that Tipin may also play a role in the intra-S-phase DNA damage checkpoint. As shown in Fig. Chou and Elledge

3E, knockdown of either ATR or Tipin by siRNA compromised the ability of those cells to down-regulate DNA synthesis after exposure to IR. This strongly suggests that Tipin is also required for the intra-S-phase DNA damage checkpoint. Loss of Tipin and Timeless Leads to Spontaneous Formation of ␥-H2AX Foci. Tipin and Timeless are likely to be components of the DNA

replication complex and may be required to stabilize forks in response to endogenous stress during every S phase. A failure to stabilize forks can lead to fork collapse and double-strand breaks (DSBs). One marker for DSBs is the accumulation of ␥-H2AX foci (28, 29). To examine roles in preventing and responding to DSBs, cells were transfected with either control or Tipin siRNAs, untreated or treated with various doses of ␥-irradiation, and analyzed for ␥-H2AX foci formation by immunofluorescence microscopy. We found that Tipin remains localized to the nucleus after DNA damage and that ␥-H2AX foci formed normally in response to DNA damage (data not shown). However, we observed a large increase in the formation of spontaneous ␥-H2AX foci in the absence of exogenous damage in both Tipin- and Timeless-depleted cells (Fig. 4A and data not shown). Notably, elevated staining of ␥-H2AX foci was found in only a subset of cells lacking Tipin by immunofluorescence. The increase in ␥-H2AX level was also confirmed by Western blotting (Fig. 4B). We interpret the above results to Tipin-depleted cells having defects in responding to endogenous replication stress. When these cells were exposed to replication stress induced by 10 mM HU, significant increases in ␥-H2AX were observed (Fig. 4B), suggesting that Tipin prevents stalled replication forks from developing into DSBs. Similar results were also observed in Timeless-depleted cells (data not shown). Overall, these results suggest that cells depleted of Tipin or Timeless may be less prepared to cope with the stress typically faced by dividing cells. Depletion of Either Tipin or Timeless Leads to the Loss of both Proteins. Our studies have shown that depletion of human Tipin

or Timeless yields very similar phenotypes. Several studies have PNAS 兩 November 28, 2006 兩 vol. 103 兩 no. 48 兩 18145

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obtained when Timeless was depleted by siRNA treatment (data not shown). Because Tipin was found to be associated with Mcm proteins, we postulated that the depletion of Tipin might also render cells sensitive to replication stress. To test this hypothesis, we again transfected cells twice within a 24-h period with the control siRNA, Tipin siRNA, or Timeless siRNA, exposed them to 2 mM HU, and determined survival. siRNA-mediated reduction of Tipin or Timeless resulted in reduced survival in response to replication stress induced by HU (Fig. 3B). Taken together, these results strongly indicate that depletion of Tipin and Timeless compromises the ability of cells to respond and survive after exposure to DNA damage and replication stress.

Fig. 4. Loss of Tipin leads to spontaneous formation of ␥-H2AX foci in the absence of exogenous damage. (A) Control or Tipin siRNA-treated HeLa cells were grown on coverslips, fixed with formaldehyde, and coimmunostained with anti-␥-H2AX and anti-Tipin antibodies. The merged picture shows cells stained with anti-␥-H2AX (green) and anti-Tipin (red) antibodies as well as DAPI (blue). (B) HeLa cells were treated with control or Tipin siRNAs and immunoblotted with anti-Tipin, anti-␥-H2AX, or anti-Ran antibodies.

also demonstrated that Tof1/Swi1 and Csm3/Swi3 mutants share very similar phenotypes in yeast (12, 14, 15, 21, 23, 24, 30). This could be explained if it is the function of the complex that is required for each of these functions. Alternatively, one protein could be the key activity, and that activity might be controlled by the other partner in the complex. To explore a potential regulatory role for Tipin in Timeless function, we examined whether depletion affected the mobility of the other component of the complex. Reduction of Tipin by each of the two siRNAs targeting Tipin led to the dramatic loss of both Tipin and Timeless proteins (Fig. 5A). We also observed that depletion of Timeless by two independent siRNAs led to the depletion of Tipin in the same population (Fig. 5B). Together, these results show that the maintenance of Timeless protein levels depends on the maintenance of Tipin protein levels and vice versa. Furthermore, it leads us to predict that the same mechanism may also function in yeast and could explain the high phenotypic similarities between cells lacking either Tof1/Swi1 or Csm3/Swi3. Discussion In this study, we characterized Tipin, to our knowledge a protein of previously undescribed function in humans. We find that Tipin and Timeless protein levels in the cell are interdependent as depletion of Tipin will lead to the depletion of Timeless in the cells and vice versa. This explains the observation that depletion of Tipin and Timeless always produce similar phenotypes but also means it will be experimentally difficult to separate the specific functions of the individual proteins. Tipin is localized predominantly to the nucleus, except in mitotic cells where the protein disperses evenly throughout the intracellular space and does not change in response to DNA damage. Furthermore, endogenous Tipin and Timeless proteins interact with each other, as well as with members of the replicative helicase, the MCM2–MCM7 complex. Importantly, 18146 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0609251103

Fig. 5. Depletion of Tipin or Timeless leads to the loss of both proteins. (A) HeLa cells were transfected with control siRNA or one of the two different siRNAs targeting Tipin. Lysates were analyzed by Western blotting with antibodies against Tipin, Timeless, or GAPDH. (B) Lysates from cells treated with control siRNA or one of the two different siRNAs targeting Timeless were analyzed by Western blotting with antibodies against Timeless, Tipin, or GAPDH.

Tipin appears to perform two major functions in human cells. First, it plays an important role in maintaining an intact DNA damage checkpoint. Loss of Tipin renders cells sensitive to both ␥-irradiation and replication stress induced by HU, with defects in both the intra-S-phase and G2/M checkpoints. This is consistent with the functions of Tipin homologs reported in yeast, as well as data from human Timeless (23, 24, 26, 30). The second function ascribed to Tipin is the suppression of endogenous DNA damage in untreated cells. Cells depleted of Tipin acquire elevated levels of ␥-H2AX, a marker for DNA DSBs. Notably, this phenotype was also recently observed in cells expressing short hairpin RNA (shRNA) targeting Claspin (31), as well as RPA siRNA-treated cells (32). Although this observation has yet to be reported in yeast, it is consistent with a role for Tipin in the cellular response to stress generated during the course of DNA replication. We speculate that loss of Tipin may lead to the accumulation of aberrant structures during DNA replication, possibly caused by failure to stabilize stalled replication forks that eventually result in DSBs. One notable caveat of this study is that we cannot rule out the possibility that the intra-S-phase checkpoint defect resulted from premature activation of the checkpoint in the absence of exogenous DNA damage. Because this assay measures the rate of DNA synthesis in ␥-irradiated cells relative to unirradiated cells, activation of this checkpoint in untreated cells would artificially deflate the perceived response to exogenous DNA damage. This caveat cannot be ignored because of the spontaneous formation of ␥-H2AX foci in the absence of Tipin and is a caveat for every treatment that causes an intra-S-phase checkpoint defect. How depletion of Tipin cripples the DNA damage checkpoint is not understood. A likely explanation is that Tipin is required for the phosphorylation of Chk1 after checkpoint activation as has been reported for Timeless (26). Because the depletion of Chou and Elledge

UACAGUGCUCCUCAUCAGAGCCUUG-3⬘), and ATR (an equal mix of the Select 3 RNAi set). Antibodies. The Tipin antibody was generated against a GST–

Tipin fusion protein at Bethyl Laboratories (Montgomery, TX). The rabbit anti-Timeless antibody was a generous gift from P. Minoo (University of Southern California, Los Angeles, CA) (34). Additional antibodies used in this study included antibodies against Mcm6 and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), ␥-H2AX and phosphohistone H3 (Upstate Biotechnology, Lake Placid, NY), Ran (BD Biosciences, Franklin Lakes, NJ), and Mcm7 (DCS-141). Radioresistant DNA Synthesis (RDS) and G2/M Checkpoint Assays.

U2OS cells were transfected with siRNAs twice within a 24-h interval for RDS assays and were processed as described previously (35). For G2/M checkpoint assays, U2OS cells were transfected with siRNAs twice within a 24-h interval and irradiated 48 h after the second transfection. Cells were harvested 1 h after irradiation, ethanol-fixed, stained with propidium iodide and anti-phosphohistone H3 antibodies followed by Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, Carlsbad, CA), and analyzed on a flow cytometer (BD Biosciences). Immunoprecipitation and Western Blot Analysis. For immunopre-

cipitations, HEK 293T cells were lysed in a buffer containing 50 mM Tris, 150 mM NaCl, and 0.5% Nonidet P-40 supplemented with protease and phosphatase inhibitors. Lysates containing 1–2 mg of protein were sonicated and incubated with Benzonase to digest cellular DNA. Insoluble material was removed by centrifugation, and the supernatants were incubated with 2 ␮g of antibodies for 1–2 h followed by protein A–Sepharose for 1 h, harvested, and immunoblotted.

Materials and Methods Plasmids and siRNA. The expression plasmid for Tipin was generated by inserting PCR-amplified Tipin cDNA in frame into pMAGIC2 (33). Stealth Select RNAi duplexes were purchased from Invitrogen (Carlsbad, CA) to down-regulate the expression of Tipin (5⬘-UUUCAGAUAAGUUUGUCAGAAAGGG-3⬘ and 5⬘-AUUUCUGGAUGUAGCAUCAAGUUGC-3⬘), Timeless (5⬘-AUAGCCCUCAGUCAUCUCUCGAACC-3⬘ and 5⬘-

We thank P. Minoo for providing the Timeless antibody; F. Stegmeier, A. Smogorzewska, R. McDonald, B. Wang, and K. Hurov for technical assistance; and members of the Elledge laboratory for helpful discussions. This work was supported by grants from the National Institutes of Health and Center for Medical Countermeasures against Radiation (to S.J.E.). S.J.E. is a Howard Hughes Medical Institute Investigator. D.M.C. is a National Science Foundation Graduate Research Fellow.

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Tipin leads to the loss of Timeless, we expected Tipin siRNAtreated cells to also have a Chk1 phosphorylation defect. However, we have been unable to either confirm or disprove this hypothesis. Although we have observed subtle defects in Chk1 phosphorylation after Tipin siRNA treatment on several occasions (data not shown), the results were not always reproducible. For example, Chk1 phosphorylation after DNA damage was reduced in several instances in Tipin siRNA-treated cells but resulted from a drop in Chk1 protein levels themselves (data not shown). We have also had difficulties detecting a Chk1 phosphorylation defect in Timeless siRNA-treated cells (data not shown). A number of factors may account for this discrepancy. One possibility is that the defect is mild and therefore difficult to detect in every single experiment. Another possibility is that cell line differences may explain the more robust DNA damage checkpoint response in our cells. We cannot rule out, for example, that a second pathway is intact in our cells that functions in parallel with Timeless and Tipin to mediate the activation of Chk1 after DNA damage. This is reminiscent of the redundant pathway in budding yeast by which Rad9 acts as a backup to activate Rad53 in the absence of Tof1 or Csm3, the respective homologs of Timeless and Tipin (7, 24). The ability to detect and reproducibly deplete Tipin should allow us to address a number of important questions in the future, such as its role in replication stress and sister chromatid cohesion, as noted for the yeast homolog (4, 18, 21, 23, 30). Furthermore, the results from our study and those from yeast (23, 30) indicate that the loss of Tipin in cells may contribute to genomic instability. Genomic instability is known to promote tumorigenesis; therefore, it will be important to determine whether Tipin or Timeless defects result in cancer.