Genes Genet. Syst. (2008) 83, p. 95–100 Vertebrate WRNIP1 and BLM are required for efficient maintenance of genome stability

Tomoko Hayashi1, Masayuki Seki1*, Eri Inoue1, Akari Yoshimura1, Yumiko Kusa1, Shusuke Tada1 and Takemi Enomoto1,2 1Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan 2Tohoku University 21st Century COE Program, “Comprehensive Research and Education Center for Planning of Drug Development and Clinical Evaluation,” Sendai, Miyagi 980-8578, Japan

(Received 25 September 2007, accepted 15 November 2007)

Bloom syndrome (BS) is rare autosomal recessive disorder associated with chro- mosomal instability. The responsible for BS, BLM, encodes a protein belonging to the RecQ family. Disruptions of the SGS1 gene of Saccha- romyces cerevisiae, which encodes the RecQ helicase homologue in the budding yeast, causes accelerated aging, and this phenotype is enhanced by the disruption of MGS1, the budding yeast homologue for WRNIP1. To examine the functional relationship between RecQ and WRNIP1 in vertebrate cells, we generated and characterized wrnip1/blm cells derived from the chicken B-lymphocyte line DT40. wrnip1/blm cells showed an additive elevation of sister chromatid exchange (SCE), suggesting that both independently contribute to the suppression of excess SCE formation. The double mutants were more sensitive to DNA damage from camptothecin (CPT), but not to damage from methyl methanesulfonate, than either single mutant. This result suggests that WRNIP1 and BLM function inde- pendently to repair DNA or induce tolerance to the lesions induced by CPT.

Key words: BLM, DT40, Mgs1, RecQ, WRN, WRNIP1

Five genes encoding RecQ helicase homologues have RecQL4 mutations result in RAPADILINO syndrome and been identified in human cells, three of which are the Baller-Gerold syndrome, suggesting that RecQL4 has mul- causative genes for Bloom syndrome (BS), Werner syn- tiple functions (Dietschy et al., 2007). drome (WS), and Rothmund-Thomson syndrome (RTS) A number of reports have shown that many cellular (Ellis et al., 1995; Yu et al., 1996; Kitao et al., 1999). BS events, including DNA repair, recombination, transcrip- is characterized by retarded growth, sunlight sensitivity, tion, telomere maintenance, and apoptosis, are affected in immunodeficiency, male infertility, and predisposition to WS cells. The gene responsible for WS encodes a protein a wide variety of malignant tumors (German, 1993), and (WRN) that possesses DNA helicase and exonuclease WS is characterized by premature aging associated with activities. It has been reported that WRN physically an early onset of age-related diseases including arterios- interacts with BLM, the protein that is defective in BS, as clerosis, malignant tumors, melituria, and cataracts well as DNA polymerase δ, replication protein A, PCNA, (Martin, 1985). WS cells show subtle defects in DNA DNA topoisomerase I (Top1), the Ku 70/86 complex, p53, replication, such as an extended S phase and reduced fre- PARP, TRF1, TRF2, and RAD52 (Kusumoto et al., quency of replication origin firing (Fujiwara et al., 1977). 2007). In addition, we found a novel WRN-interacting RTS is an autosomal recessive inherited disease charac- protein, WHIP (Werner helicase interacting protein), with – terized by premature aging, developmental abnormalities, the yeast two-hybrid method (Kawabe et al., 2001). We and predisposition to cancers such as osteogenic sarcoma have renamed WHIP as WRNIP1 (Werner Interacting (Kitao et al., 1999). Some features of RTS are reportedly Protein 1) according to the recommendation of HUGO. caused by mutations of human RecQL4 (Kitao et al., Purified WRNIP1 indeed interacts directly with WRN in 1999). Moreover, it has also been reported that distinct an ATP stimulated manner (Kawabe et al., 2006). More- over, WRNIP1 co-immunoprecipitates with WRN, suggest- Edited by Hiroshi Iwasaki ing that WRNIP1 interacts with WRN in cells (Kawabe et * Corresponding author. E-mail: [email protected] al., 2006). 96 T. HAYASHI et al.

To study the function of WRNIP1, we used budding ing with budding yeast mgs1-sgs1 cells (Branzei et al., yeast as a model system, because they possess both a 2002a). One reason for this inconsistency between ver- WRNIP1 homologue MGS1 (Maintenance of Genome tebrate cells and yeast may be the existence of multiple Stability 1) (Hishida et al., 2001), and a RECQ homo- RecQs in vertebrate cells, whereas yeast has a single logue, SGS1. MGS1 was identified as a gene that RecQ helicase, Sgs1. In addition, the human BLM gene, encodes a protein whose central region is similar to but not WRN, reportedly suppresses some of the pheno- RuvB. An mgs1 deletion mutant shows types of budding yeast sgs1 cells (Heo et al., 1999) sug- an elevated rate of mitotic recombination, which causes gesting that Sgs1 is more functionally similar to BLM genome instability, and is synthetic lethal with rad6 than WRN. In this context, we decided to examine the (Hishida et al., 2002). The Saccharomyces cerevisiae functional relationship between WRNIP1 and BLM in cells with disruptions in both mgs1 and sgs1 grow much vertebrate cells. more slowly than either single gene mutant, have a To examine the functional relationship between BLM higher frequency of recombination, and tend to arrest in and WRNIP1, we generated a double-gene-disrupted

G2/M phase, suggesting that Mgs1 and Sgs1 perform non- mutant, wrnip1/blm, from chicken DT40 cells. Since we overlapping functions to repair or otherwise accommodate had already generated wrnip1 and blm DT40 cells DNA lesions (Branzei et al., 2002a). These results (Kawabe et al., 2006; Wang et al., 2000), we generated prompted us to generate vertebrate WRNIP1-WRN dou- wrnip1/blm DT40 cells by transfecting two BLM-targeting ble-gene-knockout cells and examine their phenotypes. Although the frequency of spontaneously occurring sister chromatid exchange (SCE) in the wrnip1/wrn mutant is slightly increased compared to that of either single mutant, the double mutants grow at a rate similar to that of wild-type cells (Kawabe et al., 2006), clearly contrast-

Fig. 1. Generation of wrnip1/blm cells. DT40 cells were cul- tured in RPMI 1640 supplemented with penicillin, streptomycin, amphotericin B, 10% fetal bovine serum, and 1% chicken serum at 39.5°C. The wrnip1 and blm disruptants were generated as reported previously (Wang et al., 2000; Kawabe et al., 2006). To generate wrnip1/blm DT40 cells, we used a Gene Pulser appa- ratus (BioRad, Hercules, CA, USA) at 550 V and 25 μF to electroporate 107 wrnip1 cells with 30 μg of linearized BLM- targeting constructs (Wang et al., 2000) containing either a hygromycin or a puromycin selection marker. Drug-resistant colonies were selected in 96-well plates with medium containing 2.5 mg/ml hygromycin or 0.5 μg/ml puromycin. Gene disruption was confirmed by RT-PCR of total RNA from wild-type (lane 1), wrnip1 (lane 2), blm (lanes 3 and 4), and wrnip1/blm DT40 cells (lanes 5 [clone #1] and 6 [clone #18]). Primers used to detect WRNIP1 are as described previously (Kawabe et al., 2006). Open arrowhead and closed arrowhead indicated wild-type and Fig. 2. Growth curves and plating efficiency of wrnip1/blm the truncated form WRNIP1 mRNA, respectively, due to DT40 cells. (A) Growth curves. Cells were inoculated into 6- WRNIP1 disruption (Kawabe et al., 2006). The primers used to well plates and counted after the indicated periods. Cells that detect BLM and RECQL1 mRNA are as follows: were negative for trypan blue staining were counted as viable BLM-sense: 5’-GGTGGTAAAAGTCTGTGCTAC-3’ cells. The experiments were repeated three times, and repre- BLM-antisense: 5’-CTACAGATTTTGGAAGGGAAGC-3’ sentative data are shown. The bars indicate standard devia- RECQL1-sense: 5’-ATGACAGCTGTGGAAGTGCTAGAGGA-3’ tions. Often, the duplicate data were so close that the standard RECQL1-antisense: 5’-TCAGTCAAGAACAACAGGTTGGTCAT- deviations are hidden by the symbols. (B) Plating efficiencies of CTC-3’ cells with the indicated genotypes. Each value is the average RECQL1 was amplified as a loading control. from three independent experiments. Bars indicate standard deviations. WRNIP1 and BLM in maintenance of genome stability 97 constructs successively into wrnip1 cells. The gene dis- The most characteristic feature of BS cells is genomic ruption of WRNIP1 and BLM was verified by RT-PCR instability, which is manifested as more frequent chromo- (Fig. 1). A truncated WRNIP1 mRNA was expressed in some breaks, interchange between homologous chromo- wrnip1 and wrnip1/blm cells; our previous characteriza- somes, and SCE. Elevated SCE, a hallmark of BS, is tion indicated that the putative truncated WRNIP1 pro- thought to be an excellent indicator of replication defects. tein, if it exists in the cells, seems to have no biological Therefore, we next examined the frequency of SCE in activity (Kawabe et al., 2006). wrnip1/blm cells. As reported previously, SCE was First, we examined the proliferation of wrnip1, blm, greatly and slightly elevated in blm cells and wrnip1 and wrnip1/blm cells. Consistent with our previous cells, respectively (Fig. 3). The SCE frequency of results, wrnip1 cells grew at a rate similar to that of wild- wrnip1/blm cells was additively increased compared to type cells, whereas blm cells proliferated more slowly that of either single mutant. Since many proteins are than wild-type cells (Fig. 2A). wrnip1/blm cells prolifer- involved in the formation of SCE, and a biochemical anal- ated almost the same as blm cells. Moreover, wrnip1 ysis has shown that BLM and TOP3α dissolve double Hol- cells showed a high plating efficiency, similar to wild-type liday junctions to prevent crossover (Wu and Hickson, cells, and the plating efficiencies of wrnip1/blm cells were 2003), the elevation of spontaneous SCE in blm cells can almost the same as blm cells (Fig. 2B). be explained by the failure of the dissolution of recombi-

Fig. 3. Frequency of spontaneous SCE seen in wrnip1/blm cells. Preparation of the cells on the slides for counting spontaneously occurring sister chromatid exchanges (SCE) was described previously (Wang et al., 2000). SCE levels are shown for wild-type (A), wrnip1 (B), blm (C), and wrnip1/blm (D) cells. Numbers represent means and standard deviations of scores from 150 metaphase cells. 98 T. HAYASHI et al. nation intermediates. Previous studies of Mgs1 sug- DNA lesions during DNA replication. gested that its function is somehow related to the function We next examined the sensitivity of wrnip1/blm cells of DNA polymerase δ (Polδ), which is required for cellular to the DNA-damaging agents methyl methanesulfonate DNA replication (Hishida et al., 2002; Branzei et al., (MMS) and camptothecin (CPT) to investigate the func- 2002b). In this context, we have observed stimulation of tional relationship between WRNIP1 and BLM in the Polδ activity by WRNIP1 at the initiation stage in vitro presence of DNA damage. blm cells were moderately (Tsurimoto et al., 2005). Thus, the slight elevation of more sensitive to MMS than wild-type cells (Fig. 4A), in SCE in wrnip1 cells can be explained if lesions that agreement with the results of a previous study (Imamura remain on replicating DNA due to the defect of WRNIP1 et al., 2002). In contrast, the sensitivity of wrnip1 cells are channeled toward a homologous recombination repair to MMS was similar to that of wild-type cells (Fig. 4A), pathway that uses a sister chromatid as a template. It consistent with previous results (Kawabe et al., 2006; is likely, therefore, that the increase of spontaneous SCE Yoshimura et al., 2006), and there was little or no in wrnip1/blm cells indicates that DNA replication is enhancement of sensitivity to MMS in wrnip1/blm cells impaired in the cells lacking WRNIP1, resulting in the over that seen in blm cells. WRNIP1 thus plays no appa- production of or a failure to remove lesions that are pro- rent role in DNA repair or damage tolerance for lesions cessed by homologous recombination; then the recombina- generated by MMS, even in the absence of BLM. In con- tion intermediates are resolved to form a crossover, trast, wrnip1 cells were moderately sensitive to CPT, to resulting in SCE in the absence of BLM. These results a degree comparable to that seen in blm cells, and there coincide well with those obtained in budding yeast, was an additive increase in the sensitivity to CPT in the because a synergistic increase of spontaneous sister chro- wrnip1/blm cells (Fig. 4B). CPT is a Top1 inhibitor and matid recombination is also observed in mgs1-sgs1 double stabilizes so called “cleavage complexes,” which are mutants (Branzei et al., 2002a). Therefore, it seems formed by reversible transfer of a phosphodiester bond to likely that vertebrate WRNIP1 and BLM play roles simi- a tyrosine residue in the catalytic site of the enzyme, lar to those of their budding yeast counterparts, Mgs1 and causing single-stranded nicks (Pommier, 2004). The Sgs1, acting independently to deal with lesions during cytotoxicity of CPT is thought to be due to the DNA dou- DNA replication and consequently to decrease sister chro- ble-stranded breaks (DSBs) that are formed when repli- matid recombination. In contrast, it was reported that cation forks encounter the single-strand nicks. Thus, the disruption of the WRN gene in blm cells resulted in both WRNIP1 and BLM seem to respond to DNA lesions 2/3 reduction of SCE frequency (Imamura et al., 2002; generated by CPT, such as DSBs, but through indepen- Dong et al., 2007), suggesting that WRNIP1 and WRN dent pathways. have opposing functions in blm cells in the prevention of Genetic studies using yeast revealed that the simulta- SCE formation. Thus, although WRNIP1 binds to WRN, neous mutation of MGS1 and RAD6 (Hishida et al., 2002), WRNIP1 may act independently of WRN in dealing with RAD18 or RAD5 causes a synthetic growth defect, sug-

Fig. 4. Sensitivity of wrnip1/blm DT40 cells to MMS and CPT. Cells were treated with the indicated concentrations of MMS (A) or CPT (B). Briefly, cells (2~4 × 102) were plated into dishes containing various concentrations of methyl methanesulfonate (MMS) or camptothecin (CPT) in D-MEM/F-12 medium supplemented with 1.5% (w/v) methylcellulose, 1.5% chicken serum, and 15% fetal bovine serum. After 10 to 14 days, visible colonies were counted. The data represent the percent survival compared to untreated cells. The experiments were repeated three times, and representative data are shown. The bars indicate standard deviations. WRNIP1 and BLM in maintenance of genome stability 99 gesting that Mgs1 and Rad18/Rad5 act in a similar but Ciocci, S., Proytcheva, M., and German, J. (1995) The parallel pathway in processing stalled replication forks, Bloom’s syndrome gene product is homologous to RecQ heli- perhaps by promoting a template switching mode of DNA cases. Cell 83, 655–666. 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