Research article 2565 A Werner syndrome protein homolog affects C. elegans development, growth rate, life span and sensitivity to DNA damage by acting at a DNA damage checkpoint Se-Jin Lee, Jong-Sung Yook, Sung Min Han and Hyeon-Sook Koo* Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea *Author for correspondence (e-mail: [email protected]) Accepted 18 February 2004 Development 131, 2565-2575 Published by The Company of Biologists 2004 doi:10.1242/dev.01136 Summary A Werner syndrome protein homolog in C. elegans (WRN- irrespective of γ-irradiation, and pre-meiotic germ cells had 1) was immunolocalized to the nuclei of germ cells, an abnormal checkpoint response to DNA replication embryonic cells, and many other cells of larval and adult blockage. These observations suggest that WRN-1 acts as worms. When wrn-1 expression was inhibited by RNA a checkpoint protein for DNA damage and replication interference (RNAi), a slight reduction in C. elegans life blockage. This idea is also supported by an accelerated S span was observed, with accompanying signs of premature phase in wrn-1(RNAi) embryonic cells. wrn-1(RNAi) aging, such as earlier accumulation of lipofuscin and phenotypes similar to those of Werner syndrome, such as tissue deterioration in the head. In addition, various premature aging and short stature, suggest wrn-1-deficient developmental defects, including small, dumpy, ruptured, C. elegans as a useful model organism for Werner transparent body, growth arrest and bag of worms, were syndrome. induced by RNAi. The frequency of these defects was accentuated by γ-irradiation, implying that they were derived from spontaneous or induced DNA damage. Key words: Werner syndrome, Disease model, Aging, DNA damage, wrn-1(RNAi) worms showed accelerated larval growth Checkpoint Introduction dependent pathway is involved in the accelerated cellular Werner syndrome (WS) is associated with a rapid acceleration senescence caused by the absence of WRN. WS cells are also of aging, and is caused by mutations in the RecQ family DNA hypersensitive to certain DNA damaging agents, including the helicase gene wrn (Yu et al., 1996). wrn encodes a protein with chemical carcinogen 4-NQO (Ogburn et al., 1997), camptothecin (Poot et al., 1999), and DNA cross-linking agents a central domain of seven helicase motifs and two conserved (Poot et al., 2001). domains (RQC and HRDC) located C-terminal to the helicase WRN has been shown to form complexes with proteins domain (Morozov et al., 1997). WRN differs from other involved in cellular responses to DNA damage and in DNA members of the RecQ family in that it possesses an unusual ′→ ′ replication. The identification of a functional interaction exonuclease domain homologous to the 3 5 exonuclease between WRN and the p53 tumor suppressor protein has domain of E. coli DNA polymerase I and of RNaseD (Moser emphasized the role of the RecQ family in maintaining et al., 1997; Huang et al., 1998; Shen et al., 1998; Suzuki et genomic stability (Spillare et al., 1999). WRN also al., 1999). Nevertheless, when mutated, WRN causes genomic dramatically stimulates the cleavage reaction catalyzed by the instability disorders associated with an elevated risk of cancer, human 5′ flap endonuclease/5′→3′ exonuclease FEN1 (Brosh short stature, and/or premature aging, like two other RecQ et al., 2001), a DNA structure-specific nuclease implicated family DNA helicases in humans: the Bloom syndrome (BS) in DNA replication and repair (Lieber, 1997). The ability and the Rothmund¯Thomson syndrome (RTS, RecQL4) of replication protein A (RP-A; RPA1 – Human Gene proteins (Mohaghegh and Hickson, 2001). Nomenclature Database) to stimulate the unwinding of long Several defects at the cellular level have been detected in stretches of DNA duplex by WRN helicase suggests that WRN WS. Cells cultured from WS patients have a reduced life span, may function in replication, a notion supported by interaction an extended S phase, and reduced RNA transcription by RNA of WRN with other replication proteins. Recent evidence polymerases I and II (Martin et al., 1970; Salk et al., 1985; points to a direct protein interaction between WRN and the Balajee et al., 1999; Shiratori et al., 2002). Although no direct Ku80/70 heterodimer implicated in non-homologous end- role of WRN has been established in telomere metabolism, WS joining of double-strand breaks (Cooper et al., 2000). In fibroblasts expressing a transfected human telomerase (TERT) addition to these functional interactions, WRN has been gene have an increased life span and can be immortalized reported to physically interact with human polymerase delta (Wyllie et al., 2000). These results suggest that a telomerase- (Szekely et al., 2000), PCNA and DNA topoisomerase I (Label 2566 Development 131 (11) Research article et al., 1999). These interactions suggest that WRN is a central The overexpressed protein band was excised, crushed in PBS, mixed player in a macromolecular complex essential for DNA with Freund’s adjuvant, and then injected into Balb/c mice four times replication or repair. at weekly intervals (200 µg protein per injection). In C. elegans, four RecQ family proteins are predicted from Immunostaining the genomic DNA sequence. Of these proteins, the one encoded by the open reading frame (ORF) F18C5.2 is most C. elegans embryos were immunostained by a slightly modified version of the procedure of Crittenden and Kimble (Crittenden and homologous to human WRN. To understand the role of this C. Kimble, 1999). Embryos were freeze-cracked, fixed, incubated with elegans WRN homolog (WRN-1), we localized the protein in polyclonal mouse antiserum against the N-terminal 209 amino acids C. elegans and investigated the phenotypes arising from of WRN-1 (1:25 dilution), and then with FITC-conjugated goat anti- inhibited expression. mouse immunoglobulin G (1:500 dilution, Santa Cruz Biotechnology) pre-treated with C. elegans acetone powder. After being stained with Materials and methods DAPI (4,6-diamidino-2-phenylindole, 1 µg/ml), specimens were observed with a fluorescence microscope (DMR HC, Leica). Gonads Materials and intestines were extruded by decapitating adult C. elegans, fixed Bristol N2, as a standard wild-type strain, and div-1(or148ts) strains in 3% paraformaldehyde, then immunostained as described (Jones et were obtained from the C. elegans Genetics Center (St Paul, MN, al., 1996). Whole-worm staining was carried out by the collagenase USA). An EST clone of the Ce-wrn-1 gene (yk41c3) was provided method of Nonet et al. (Nonet et al., 1993). After fixation in 4% by Dr Y. Kohara (National Institute of Genetics, Japan). paraformaldehyde, worms were incubated in a reducing solution [5% Deoxynucleotide oligomers were synthesized at Genotech (Korea). β-mercaptoethanol, 1% Triton X-100, 0.1 M Tris-Cl (pH 6.9)] at 37°C overnight, and then reacted with collagenase (1000 units/ml, Sigma) ′ Cloning of a 5 cDNA fragment of C. elegans wrn-1 by RT- in buffer [0.1 M Tris-Cl (pH 7.5), 1 mM CaCl2] at 37°C for 5 hours. PCR and construction of full-length cDNA Subsequent reactions with primary and secondary antibodies were as The EST clone yk41c3 of the F18C5.2 ORF lacked the first exon (115 described above for embryos. nucleotides; nt) of the predicted ORF of 16 exons. Therefore, to obtain a 5′-terminal cDNA clone, we isolated C. elegans total RNA using Inhibition of wrn-1 expression by double-stranded RNA an RNeasy kit (Qiagen). cDNA synthesis progressed in a reaction microinjection mixture (50 µl) containing C. elegans total RNA (3 µg), a primer The pCeWRN recombinant plasmid was linearized with BamHI and (10 pmoles) of sequence 5′-GTGGACATAAGAACAAATTGGTC-3′ ApaI restriction enzymes at its multicloning site to prepare antisense (nt 752-729 in the ORF) from exon 3, and Superscript reverse and sense transcripts of wrn-1, respectively. Antisense RNA was transcriptase II (200 units, Stratagene), at 42°C for 1 hour. First cDNA synthesized using BamHI-digested plasmid DNA (2 µg), T7 RNA strand synthesis was terminated by heating at 70°C, and then template polymerase (5 units, MBI), ribonucleoside triphosphates (rNTPs, 0.4 RNA was degraded by RNase H (2 units, Takara). A cDNA fragment mM each) and RNase inhibitor (5 units, Takara) in buffer [40 mM Tris- was amplified from the first cDNA strand by PCR (polymerase chain Cl (pH 8.0), 8 mM MgCl2, 2 mM spermidine, 50 mM NaCl, 18 mM reaction) using the SL1 primer (5′-GGTTTAATTACCCAAGT- DTT; total 50 µl], at 37°C for 2 hours. Sense RNA was synthesized TTGAG-3′) and a primer of sequence 5′-CATTTCTGACAACATCC- under the same reaction conditions as described for antisense RNA, CACTG-3′ (nt 715-694 in the ORF) from exon 3. The amplified except for the use of ApaI-treated DNA (2 µl) and T3 RNA polymerase cDNA fragment was cloned into pGEM-T vector (Promega) and (5 units, MBI). After RNA synthesis, RNase-free DNase I (2 units) sequenced with an ABI PRISM Dye Terminator Cycle Sequencing was added to degrade template DNA, and then phenol (pH 4.5) Ready Reaction Kit (Perkin-Elmer). extraction and ethanol precipitation were carried out. An equivalent To obtain a full-length cDNA construct of wrn-1, PCR was carried amount of sense and antisense RNAs were mixed to a total out using two primers: 5′-CGCGGATCCATGATAAGTGATGAT- concentration of 1 µg/µl, and then microinjected into the intestines of GACGATC-3′, containing nt 1-22 of the ORF, and a BamHI young adult N2 worms. The worms were placed on an NGM plate with recognition sequence (underlined); and 5′-CATTTCTGACAACATC- an E. coli OP50 lawn, and were transferred to new plates after 12 hours. CCACTG-3′, corresponding to nt 715-694 of the ORF.