Biochimica et Biophysica Acta 1813 (2011) 473–479

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The N-terminal region of RECQL4 lacking the helicase domain is both essential and sufficient for the viability of vertebrate cells Role of the N-terminal region of RECQL4 in cells

Takuya Abe a, Akari Yoshimura a,b, Yoshifumi Hosono a, Shusuke Tada a,c, Masayuki Seki a, Takemi Enomoto a,b,⁎ a Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan b Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi Tokyo 202-8585, Japan c Faculty of Pharmaceutical Sciences, Teikyo Heisei University, 4-1 Uruido Minami, Ichihara, Chiba 290-0193, Japan article info abstract

Article history: Rothmund–Thomson syndrome (RTS) is a rare genetic disorder characterized by premature aging, Received 24 June 2010 developmental abnormalities, and a predisposition to cancer. RTS is caused by mutations in the RECQL4 Received in revised form 2 December 2010 gene, which encodes one of the five human RecQ helicases. To identify the cellular functions of RECQL4, we Accepted 3 January 2011 generated a chicken DT40 cell line in which RECQL4 expression could be turned off by doxycycline (Dox). Available online 20 January 2011 Upon exposure to Dox, cells stopped growing and underwent apoptosis. The cells could be rescued by – Keywords: expression of the N-terminal region of RECQL4 (amino acids 1 496), which lacks the helicase domain and has RecQL4 sequence similarity to yeast Sld2, which plays an essential function in the initiation of DNA replication in Rothmund–Thomson syndrome Saccharomyces cerevisiae. Smaller fragments of the N-terminal region of RECQL4 did not rescue the cells from RecQ helicase lethality. RECQL4 gene knockout cells complemented with RECQL4 (1–496) showed relatively high sensitivity DNA repair to DNA damaging agents that induce double strand breaks and cross-links, suggesting that the C-terminal Sld2 region including the helicase domain of RECQL4 is involved in the repair of certain types of DNA lesions. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Gerold syndrome, both of which have features that overlap but there are also some that differ from those of RTS [8,9]. The RecQ family of DNA helicases is highly conserved throughout Thus far, three different RECQL4 knockout mice have been evolution and plays an important role in the maintenance of genomic reported. One of these knockout mice was embryonic lethal [10]. stability. Higher eukaryotic cells possess five RecQ family helicases. Another was generated by deleting exon 13, which encodes part of the Mutations in three of the five human helicase genes (BLM, WRN, and central RecQ-helicase domain. However, although 5% of the mice RECQL4) are responsible for Bloom syndrome, Werner syndrome, and survived up to 14 days, they failed to develop any malignancies, a Rothmund–Thomson syndrome (RTS), respectively [1–3], that are unique characteristic of RTS [11]. The third knockout mouse was characterized by a predisposition to cancer and premature aging. generated by deleting exons 9–13. These mice survived to adulthood Recently, human RECQL4 is proved to have DNA helicase activity with some exhibiting the typical clinical features of RTS [12]. in vitro [4,5]. RTS displays a heterogeneous clinical profile that in- The involvement of RECQL4 in DNA replication was revealed in cludes growth deficiency, photosensitivity with poikiloderma, cata- studies using Xenopus egg extracts. One study showed that immuno- racts, early graying and loss of hair, and a predisposition to cancer depletion of RECQL4 from Xenopus egg extracts caused severe defects particularly osteosarcoma [6]. Mutations in the RECQL4 gene are the in the initiation of DNA replication [13]. Another study showed that cause of 60% of RTS cases [7]. Therefore, RTS is a heterogeneous the N-terminal region of RECQL4 was sufficient for the initiation of disease and mutations in other genes appear to be responsible for the DNA replication [14]. This N-terminal region of RECQL4 has sequence phenotypes seen in the remaining RTS patients. However, RECQL4 is similarity to yeast Sld2, which plays an essential role in the initiation also known to be responsible for RAPADILINO syndrome and Baller– of DNA replication in S. cerevisiae [15]. Recently, in human cells, RECQL4 has been shown to associate with DNA replication origins in a cell cycle-regulated fashion, to associate with Mcm2-7 helicase, and to be involved in the assembly of the Cdc45-Mcm2-7-GINS complex ⁎ Corresponding author. Molecular Cell Biology Laboratory, Research Institute of [16,17].InDrosophila, RECQL4 is also required for DNA replication and Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi Tokyo 202-8585, Japan. Tel./fax: +81 42 468 8645. its DNA helicase activity is essential for viability [18,19]. In addition to E-mail address: [email protected] (T. Enomoto). DNA replication, RECQL4 appears to be implicated in some types of

0167-4889/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2011.01.001 474 T. Abe et al. / Biochimica et Biophysica Acta 1813 (2011) 473–479

DNA repair, such as double strand break repair [20–22], base excision appropriate primers on a hRECQL4 cDNA template and inserted into repair [23], and nucleotide excision repair [24], and cells from RTS the vector, pEGFP-C1. The hRECQL4 (K508A) mutant was prepared patients actually show relatively high sensitivity to some genotoxic using a QuickChange site-directed mutagenesis kit (Stratagene). agents [25,26]. However, the role played by RECQL4 in DNA repair Cells expressing mutant GFP-hRECQL4 were obtained by subsequent pathways has not been clarified. transfection of these vectors into RECQL4−/−/− +FLAG-hRECQL4 The data reported so far seem to indicate that the essential function cells. To generate RECQL4−/−/− +hRECQL4 (1–496) cells, DT40 cells of RECQL4 is carried by the N-terminal region and that the DNA helicase were transfected with puro-RECQL4,his-RECQL4,hRECQL4 (1–496)/ activity of RECQL4 is somehow involved in DNA repair. In this study, to pEGFP-C1, and bsr-RECQL4, in that order. investigate the role of RECQL4 (especially the N-terminal region of RECQL4) in supporting cell viability, we generated conditional RECQL4- 2.3. Detection of apoptosis deficient cells expressing different sized fragments of RECQL4. The results indicate that the N-terminal region of RECQL4 lacking the DNA Cells were fixed in 4% formaldehyde for 15 min and stained with helicase domain is sufficient to support viability. Also, the data suggest 10 μg/ml Hoechst 33258 for 5 min. Cells with a fragmented nucleus that the C-terminal region including the DNA helicase domain of were defined as apoptotic. RECQL4 is involved in the repair of certain types of DNA lesion. 2.4. Cell cycle analysis using flow cytometry 2. Experimental procedures Flow cytometric analysis was carried out as previously described 2.1. Cell culture [28]. Briefly, cells were fixed in 70% ethanol, stained with propidium iodide (PI), and then analyzed by flow cytometry. Cells were cultured at 39 °C in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% chicken serum, and 100 μg/ml 2.5. Western blotting kanamycin in the presence or absence of 1 μg/ml doxycycline (Dox). Western blotting was carried out as previously described [29] using 2.2. Construction of plasmids and gene disruption antibodies against β-actin (Sigma), FLAG-M2 (Sigma), and γ-H2AX (Upstate) followed by a horseradish peroxidase-conjugated anti-mouse Three RECQL4 disruption constructs were generated from IgG secondary antibody (Cell Signaling). Bands were visualized using genomic PCR products inserted into a puromycin (puro), histidinol ECL detection reagents (Amersham Pharmacia). (his), or blasticidin (bsr) selection marker cassette. Human RECQL4 (hRECQL4) cDNA was prepared by RT-PCR and a FLAG tag was added 2.6. Assay for sensitivity to DNA damaging agents to the C-terminus by PCR. The FLAG-hRECQL4 was inserted into an expression vector carrying a tet-repressible promoter, pUHG 10-3 To determine drug sensitivity, 1×104 cells were inoculated into [27]. DT40 cells were successively transfected with puro-RECQL4, 24-well plates containing various concentrations of DNA damaging FLAG-hRECQL4/pUHG 10-3, his-RECQL4 and bsr-RECQL4 to generate agents in 1 ml of medium. Cells were counted after 48 h and the RECQL4−/−/− +FLAG-hRECQL4 cells. To prepare the deletion percentage survival was determined by taking the number of mutants of hRECQL4, mutant DNAs were prepared by PCR using untreated cells as 100%.

Fig. 1. Generation of RECQL4−/−/− +FLAG-hRECQL4 cells. (A) Schematic representation of the DT40 RECQL4 locus and gene targeting constructs. Closed boxes indicate exons. (B) Strategy for gene disruption. (C) Suppression of hRECQL4 mRNA expression in RECQL4–/–/– +FLAG-hRECQL4 (Q4−/−/− +FLAG-hQ4) cells by doxycycline (Dox). RNA was prepared from RECQL4−/−/− +FLAG-hQ4 cells cultured in the presence of Dox for the indicated periods. cRECQL4 cDNA was prepared by RT-PCR and electrophoresed in a 2% agarose gel. The β-actin was used as the control. (D) Disappearance of the FLAG-hRECQL4 protein during cell culture. Whole-cell lysates were prepared from Q4−/−/− +FLAG-hQ4 cells cultured in the presence of Dox for the indicated periods. FLAG-RECQL4 and β-actin (loading control) were detected by western blotting. Asterisk indicates non-specificband. T. Abe et al. / Biochimica et Biophysica Acta 1813 (2011) 473–479 475

hRECQL4 cells. Finally, after transfection of the bsr-RECQL4 construct, we obtained RECQL4−/−/− +FLAG-hRECQL4 cells. Disruption of the RECQL4 gene was confirmed by RT-PCR (Fig. 1C). Addition of Dox to RECQL4−/−/− +FLAG-hRECQL4 cells suppressed the expression of FLAG- hRECQL4 and the protein disappeared within 24 h (Fig. 1D).

3.2. Depletion of RECQL4 results in cell death

To study the effects of RECQL4 depletion on cell growth, RECQL4−/−/− +FLAG-hRECQL4 cells were grown in the presence of Dox. RECQL4−/−/− +FLAG-hRECQL4 cells stopped growing within 72 h after the addition of Dox (Fig. 2A). Flow cytometric analysis revealed little change in the cell cycle distribution pattern up to 48 h although a slight increase in G2 fraction was observed at 48 h (Supplemental Fig. 1). However, the sub-G1 fraction corresponding to dead cells, was increased at 72 h and further increased at 96 h. Importantly, the percentage of apoptotic cells, characterized by nuclear fragmentation and the level of γ-H2AX [29], increased gradually; the increase in γ-H2AX was apparent even at 24 h (Fig. 2B and C). These results indicate that RECQL4 is essential for cell viability and that chicken RECQL4 can be replaced by human RECQL4.

3.3. The N-terminal domain of RECQL4 is sufficient for cell viability

RECQL4 contains a Sld2 homology domain within its N-terminal region and a helicase domain conserved among RecQ family proteins in its middle region. RTS patients carry mutated forms of RECQL4 and almost all mutations are found in the middle or C-terminal regions of RECQL4, while the N-terminal region remains intact [8]. To examine the importance of the N-terminal region and the DNA helicase domain for cell viability, we constructed two mutants expressing GFP- conjugated hRECQL4 (K508A) and hRECQL4 (1–496) from the parent RECQL4−/−/− +FLAG-hRECQL4 cells (Fig. 3A). The substitution of K508 in the Walker A motif of RECQL4 is reported to abolish the helicase activity derived from the helicase domain of RECQL4 [4]. – – Fig. 2. Induction of cell death in the absence of RECQL4. (A) Growth curve. Cells hRECQL4 (1 496) lacks amino acids 497 1208 of hRECQL4, which (1×105)wereinoculatedin1mlmediumandculturedfortheindicatedperiodsinthe include the helicase domain and the C-terminal region. Cells presence or absence of Dox. (B) Apoptosis indicated by karyopyknosis. Upper panel: expressing GFP-conjugated full-length hRECQL4 were also generated, stained images of nuclei. Cells were fixed and stained with Hoechst 33258. The arrows and the growth of these cells was examined in the presence or indicate karyopyknosis. Lower panel: induction of apoptosis monitored by karyo- absence of Dox. Expression levels of GFP-hRECQL4 and various pyknosis. (C) Apoptosis indicated by the increase of γ-H2AX. Whole-cell lysates were − − − fl prepared from Q4 / / +FLAG-hQ4 cells cultured in the presence of Dox for the mutants of GFP-hRECQL4 were also monitored by ow cytometry indicated periods. γ-H2AX, FLAG-RECQL4 and β-actin (loading control) were detected (Supplemental Fig. 2). Although all mutant cells grew marginally by western blotting. Asterisk indicates non-specificband. more slowly than wild-type cells (Fig. 3B, left panel), all mutant cells except RECQL4−/−/− +FLAG-hRECQL4 cells were able to grow in the 3. Results presence of Dox (Fig. 3B, right panel). These results clearly indicate that the helicase domain of RECQL4 is not necessary for cell viability. 3.1. Generation of conditional RECQL4-deficient cells To determine the regions of RECQL4 that are essential for viability, the region spanning amino acids 1–496 was divided into four parts. To confirm that RECQL4 is essential for cell viability, we generated Fragments corresponding to each of the four parts, or fragments chicken DT40 cells in which the expression of the RECQL4 gene could lacking one or two parts, were then expressed (Fig. 4A). Cells be turned off by Dox treatment. The genome of DT40 cells consists of expressing the truncated N-terminal region of RECQL4 were not able macro-chromosomes 1–5, the sex chromosome Z, and many other to grow in the presence of Dox (Fig. 4B, panels b, d, and f), suggesting – micro-chromosomes. Using a database of chicken DNA sequences that almost the entire 1 496 region is required for viability. It is worth – (NW_001485527.1), the chicken RECQL4 gene was located on one of noting that cells expressing the N1 2 fragment stopped growing −/−/− the micro-chromosomes, and this chromosome later turned out to be earlier than RECQL4 +FLAG-hRECQL4 cells (Fig. 4B, panel f), – a trisomy. Three gene targeting constructs were designed to replace suggesting that N1 2 has a dominant-negative function under part of the chicken RECQL4 gene, corresponding to exons 12–13 in conditions of low RECQL4 expression due to the presence of Dox. mice and exon 13 in humans, with puro, his, or bsr selection marker cassettes (Fig. 1A). 3.4. The C-terminal region including the helicase domain of RECQL4 is After disruption of the first RECQL4 allele with the puro-RECQL4 implicated in DNA repair construct (Fig. 1B), a FLAG tag-conjugated human RECQL4 (FLAG- hRECQL4) gene linked downstream of a tet-repressible promoter was To investigate whether the C-terminal region including the helicase transfected into the cells. RECQL4+/+/− cells expressing FLAG-hRECQL4 domain of RECQL4 is involved in DNA repair, cells continuously (RECQL4+/+/− +FLAG-hRECQL4 cells) were selected, and these cells expressing the 1–496 region of hRECQL4 were generated (Fig. 5A). were transfected with his-RECQL4 to yield RECQL4+/−/− +FLAG- Although the RECQL4−/−/− +GFP-hRECQL4 (1–496) cells grew more 476 T. Abe et al. / Biochimica et Biophysica Acta 1813 (2011) 473–479

Fig. 3. DNA helicase domain of RECQL4 is not required for viability. (A) Schematic representation of mutant proteins. GFP-hQ4; GFP conjugated full length human RECQL4, GFP- hQ4 (K508A); GFP-conjugated human RECQL4 carrying a mutation of K508A, GFP-hQ4 (1–496A) ; GFP-conjugated N terminal region of RECQL4 (1–496). (B), (C) Growth curves. Q4−/−/− +FLAG-hQ4 cells (1×105)expressingGFP-Q4(1–496), GFP-Q4 (K508A), or GFP-Q4 were inoculated and cultured for the indicated periods in the presence or absence of Dox. Expression levels of GFP-hRECQL4 and various mutants of GFP-hRECQL4 were monitored by flow cytometry (Supplemental Fig. 2). slowly than the wild-type cells, their cell cycle distribution pattern was In contrast, DNA helicase activity of RECQL4 appears to be essential comparable to that of the wild-type cells (Fig. 5A, panels b and c). for viability in Drosophila because DNA helicase-deficient RECQL4 is To examine the role of the C-terminal region including the helicase not able to rescue the viability of null mutant flies [19]. This domain of RECQL4 in DNA repair, the sensitivity of RECQL4−/−/− + discrepancy may be explained by the fact that fruit flies have only GFP-hRECQL4 (1–496) cells to various DNA damaging agents was three RecQ helicases, BLM, RECQL4, and RECQL5, while chickens have compared with that of wild-type cells. RECQL4−/−/− +GFP-hRECQL4 five. Thus, the RECQL4 DNA helicase activity required for viability in (1–496) cells showed marginally increased sensitivity to methyl fruit flies is compensated for in vertebrate cells by other RecQ methanesulfonate, mitomycin C, cisplatin, and etoposide compared helicases. with wild-type cells, but were not more sensitive to 4-nitroquinoline RECQL4−/−/− +FLAG-hRECQL4 cells grew relatively normally for 1-oxide, camptothecin and hydroxyurea (Fig. 5B). This suggests that 48 h in the presence of Dox, although FLAG-hRECQL4 disappeared the C-terminal region including the helicase domain of RECQL4 is within 24 h (Fig. 1C). Considering that the doubling time of DT40 cells involved in the repair of some types of DNA lesion. is about 8 h, the cells divided at least twice in the absence of RECQL4. If RECQL4 functions to initiate DNA replication, as shown in many 4. Discussion systems [13,14,16–18], why are RECQL4-depleted cells able to initiate DNA replication in the absence of RECQL4? One of the possibilities to The results presented in this study clearly demonstrate that explain the discrepancy is that the amount of RECQL4 required for the RECQL4 is essential for the viability of vertebrate cells. The lethal essential function is very low, and a trace of RECQL4 not detectable by phenotype of RECQL4−/−/− +FLAG-hRECQL4 cells in the presence of western blotting performs this function. In this context, we cannot Dox was rescued by the expression of hRECQL4 (1–496) or hRECQL4 exclude the possibility that a trace amount of N-terminal region of (K508A), suggesting that the Sld2-like N-terminal domain of RECQL4 chicken RECQL4 is expressed in the RECQL4-depleted cells because is sufficient for cell viability and that the helicase domain of RECQL4 is chicken RECQL4 genes encoding for the N-terminal region of RECQL4 not necessary for viability. Recently, the Sld2-like N-terminal domain are left in the RECQL4 gene disrupted cells. However, the expression of of hRECQL4 was shown to possess ATP-dependent DNA unwinding N-terminal region of chicken RECQL4 seems to be very low, if any, activity in vitro [4]. because the RECQL4 gene disrupted cells die by suppressing T. Abe et al. / Biochimica et Biophysica Acta 1813 (2011) 473–479 477

Fig. 4. Dissection of the N-terminal region of RECQL4 (1–496). (A) Schematic representation of mutant proteins. (B) Growth curves. Q4−/−/− +FLAG-hQ4 cells (1×105) expressing each mutant protein were inoculated and cultured for the indicated periods in the presence or absence of Dox. Expression levels of GFP-hRECQL4 and various mutants of GFP- hRECQL4 were monitored by flow cytometry (Supplemental Fig. 2).

expression of exogenous hRECQL4. This issue must be addressed in sooner than RECQL4−/−/− +FLAG-hRECQL4 cells. This suggests that the future study. hRECQL4 (1–196) has a dominant-negative function under conditions To identify the precise region of RECQL4 essential for viability, we of reduced hRECQL4 expression and that an important region exists dissected the N-terminal region of hRECQL4 (1–496). Unfortunately, within this fragment. we were unable to narrow down the essential region because none of Many of the mutations confirmed in RECQL4 of RTS patients are the fragments tested supported viability. On the contrary, in the located in the helicase domain, or within the C-terminal region. This presence of Dox, cells expressing hRECQL4 (1–196) stopped growing indicates that loss of helicase activity may contribute to the symptoms 478 T. Abe et al. / Biochimica et Biophysica Acta 1813 (2011) 473–479

Fig. 5. Sensitivity of RECQL4−/−/− +GFP-hRECQL4 (1–496) cells to various agents. (A) Generation of RECQL4−/−/− +GFP-hRECQL4 (1–496) (Q4−/−/− +FLAG-hQ4 (1–496)) cells. a, Schematic representation of the strategy used to generate Q4−/−/− +FLAG-hQ4 (1–496) cells; b, growth curve; c, FACS analysis. (B) Assay for sensitivity to various agents. Cells were cultured in the presence or absence of DNA damaging agents for 48 h. The number of living cells was then counted. a, camptothecin (CPT); b, hydroxyurea (HU); c, 4-nitroquinoline 1-oxide (4NQO); d, methyl methanesulfonate (MMS); e, etoposide; f, cisplatin (CDDP); and g, mitomycin C (MMC).

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