Title: The MRT-1 nuclease is required for DNA crosslink repair and telomerase activity in vivo in Caenorhabditis elegans
Abstract: Article24 September 2009free access The MRT-1 nuclease is required for DNA crosslink repair and telomerase activity in vivo in Caenorhabditis elegans Bettina Meier Bettina Meier Department of Genetics, University of North Carolina, Chapel Hill, NC, USA Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, UK Search for more papers by this author Louise J Barber Louise J Barber DNA Damage Response Laboratory, Cancer Research UK, Clare Hall Laboratories, South Mimms, UK Search for more papers by this author Yan Liu Yan Liu Department of Genetics, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Ludmila Shtessel Ludmila Shtessel Department of Genetics, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Simon J Boulton Simon J Boulton DNA Damage Response Laboratory, Cancer Research UK, Clare Hall Laboratories, South Mimms, UK Search for more papers by this author Anton Gartner Anton Gartner Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, UK Search for more papers by this author Shawn Ahmed Corresponding Author Shawn Ahmed Department of Genetics, University of North Carolina, Chapel Hill, NC, USA Department of Biology, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Bettina Meier Bettina Meier Department of Genetics, University of North Carolina, Chapel Hill, NC, USA Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, UK Search for more papers by this author Louise J Barber Louise J Barber DNA Damage Response Laboratory, Cancer Research UK, Clare Hall Laboratories, South Mimms, UK Search for more papers by this author Yan Liu Yan Liu Department of Genetics, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Ludmila Shtessel Ludmila Shtessel Department of Genetics, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Simon J Boulton Simon J Boulton DNA Damage Response Laboratory, Cancer Research UK, Clare Hall Laboratories, South Mimms, UK Search for more papers by this author Anton Gartner Anton Gartner Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, UK Search for more papers by this author Shawn Ahmed Corresponding Author Shawn Ahmed Department of Genetics, University of North Carolina, Chapel Hill, NC, USA Department of Biology, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Author Information Bettina Meier1,2, Louise J Barber3, Yan Liu1, Ludmila Shtessel1, Simon J Boulton3, Anton Gartner2,‡ and Shawn Ahmed 1,4,‡ 1Department of Genetics, University of North Carolina, Chapel Hill, NC, USA 2Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, UK 3DNA Damage Response Laboratory, Cancer Research UK, Clare Hall Laboratories, South Mimms, UK 4Department of Biology, University of North Carolina, Chapel Hill, NC, USA ‡These authors contributed equally as senior authors to this work *Corresponding author. Department of Genetics, University of North Carolina, Coker Hall, Chapel Hill, NC 27599-3280, USA. Tel.: +1 919 843 4780; Fax: +1 919 962 4296; E-mail: [email protected] The EMBO Journal (2009)28:3549-3563https://doi.org/10.1038/emboj.2009.278 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The telomerase reverse transcriptase adds de novo DNA repeats to chromosome termini. Here we define Caenorhabditis elegans MRT-1 as a novel factor required for telomerase-mediated telomere replication and the DNA-damage response. MRT-1 is composed of an N-terminal domain homologous to the second OB-fold of POT1 telomere-binding proteins and a C-terminal SNM1 family nuclease domain, which confer single-strand DNA-binding and processive 3′-to-5′ exonuclease activity, respectively. Furthermore, telomerase activity in vivo depends on a functional MRT-1 OB-fold. We show that MRT-1 acts in the same telomere replication pathway as telomerase and the 9-1-1 DNA-damage response complex. MRT-1 is dispensable for DNA double-strand break repair, but functions with the 9-1-1 complex to promote DNA interstrand cross-link (ICL) repair. Our data reveal MRT-1 as a dual-domain protein required for telomerase function and ICL repair, which raises the possibility that telomeres and ICL lesions may share a common feature that plays a critical role in de novo telomere repeat addition. Introduction The ends of linear chromosomes, telomeres, pose two major challenges to the maintenance of chromosome integrity and overall genome stability: telomeres need to be adequately replicated, to compensate for the inability of canonical DNA polymerases to replicate the chromosome terminus, and they need to be protected from being mistakenly sensed and repaired as DNA double-strand breaks (DSBs). Telomeric DNA is composed of simple, repetitive sequences. The 5′-to-3′ telomeric DNA strand is G-rich and terminates as a 3′ single-stranded overhang. Telomeric repeats are replenished by telomerase, a ribonucleoprotein composed of the telomerase reverse transcriptase (TERT) and an RNA component (TR) containing the telomere-repeat template (Greider and Blackburn, 1989; Collins, 2006). The catalytic subunit of telomerase, TERT, and its RNA component are sufficient to confer telomere-repeat addition in vitro. Additional proteins, many of which are essential, facilitate processing of TR and telomerase holoenzyme function (Collins, 2006; Fu and Collins, 2007; Venteicher et al, 2008). Mutations in the essential proteins dyskerin, NOP10, NHP2, as well as in TR and TERT, confer shortened telomeres and reduced in vitro telomerase activity in patients with heritable forms of Dyskeratosis Congenita and Pulmonary Fibrosis (reviewed by Vulliamy and Dokal, 2008). Thus, telomere maintenance defects can limit proliferation of cells in lymphatic or pulmonary systems in vivo, consistent with evidence that telomerase can limit the proliferative lifespan of human primary cells in vitro (Garcia et al, 2007). Double mutants deficient for fission or budding yeast DNA-damage sensor proteins ATM and ATR display progressive telomere erosion, suggesting that DNA-damage signalling may be required for telomerase-mediated telomere maintenance in vivo (Naito et al, 1998; Ritchie et al, 1999; Nakamura et al, 2002). Conceptually related results were reported for Caenorhabditis elegans DNA-damage response mutants, where telomerase-mediated telomere replication was abolished in vivo by single mutations in subunits of the Rad9-Rad1-Hus1 (9-1-1) PCNA-like sliding clamp, mrt-2 (the worm rad1) or hus-1, or its large RFC clamp loader subunit, hpr-17 (Ahmed and Hodgkin, 2000; Hofmann et al, 2002; Boerckel et al, 2007). Further, subunits of the homologous mammalian 9-1-1 complex, as well as its RFC clamp loader, RAD17, were shown to physically interact with the telomerase holoenzyme, to bind to telomeric DNA in vivo, and to facilitate telomerase activity in vitro (Francia et al, 2006). However, knockdown of these mammalian DNA-damage response proteins is cell-lethal and results in dramatic, rapid effects on telomere length, precluding analysis of effects on telomerase-mediated telomere length maintenance in vivo (Francia et al, 2006). The former results suggest that DNA-damage response proteins may function at chromosome ends as a prerequisite for telomere repeat addition by telomerase. These proteins can respond to DSBs (d'Adda di Fagagna et al, 2004), suggesting that telomeres may be sensed as 'aberrant' DSBs when they are replicated during S-phase and that the 9-1-1 complex in conjunction with its clamp loader may facilitate recruitment of telomerase to chromosome termini. Here we identify C. elegans MRT-1 as a novel factor required for in vivo telomerase activity. MRT-1 encodes a dual-domain protein with an N-terminus homologous to the second OB DNA-binding fold found in POT1 (Protection Of Telomeres 1) proteins and a C-terminus bearing homology to the SNM1 nuclease family. SNM1 proteins function in DNA repair and checkpoint responses to interstrand cross-links (ICLs), stalled replication forks and DSBs, as well as in telomere protection (Henriques and Moustacchi, 1980; Dronkert et al, 2000; Demuth et al, 2004; Jeggo and Lobrich, 2005; Freibaum and Counter, 2006; Lenain et al, 2006; van Overbeek and de Lange, 2006; Bae et al, 2008; Hazrati et al, 2008; Hemphill et al, 2008). Although previously described C. elegans DNA-damage response mutants that are deficient for telomerase activity in vivo are hypersensitive to DSBs and ICLs, mrt-1 mutants are only deficient for ICL repair. Thus, MRT-1 defines a dual-domain ICL DNA-damage response protein that may process and interact with chromosome termini prior to telomerase-mediated telomere repeat addition. Results MRT-1 is required for telomerase activity in vivo To identify non-essential mutations that compromise telomerase activity in vivo in C. elegans, genetic screens for ethylmethanesulphonate (EMS)-induced mortal germline (mrt) mutations that resulted in progressive telomere shortening and progressive sterility, accompanied by telomere–telomere fusions, were performed. Two alleles of mrt-1, e2661 and yp2, were identified in such screens (Y Liu and S Ahmed, unpublished data) (Ahmed and Hodgkin, 2000). mrt-1 mutants showed progressive reduction in progeny and eventual sterility comparable to mrt-2 mutants and mutants defective for the C. elegans catalytic subunit of telomerase, trt-1, (Table I) accompanied by progressive telomere shortening over successive generations (Figure 1A and Figure 5). Although telomere length does fluctuate as N2 wild-type strains are propagated for multiple generations (Ahmed et al, 2001), wild-type telomeres appeared as diffuse bands on Southern blots, whereas telomeres of all mrt-1 alleles appeared as discrete bands that shorten progressively (Figure 1A, right panel), as previously observed for C. elegans mutants that are deficient for telomerase mediated-telomere replication such as trt-1, mrt-2, hus-1 and hpr-17 (Ahmed and Hodgkin, 2000; Hofmann et al, 2002; Meier et al, 2006; Boerckel et al, 2007). In crosses between mrt-1(e2661) and wild-type, mrt-1−/− F2 siblings displayed telomere erosion accompanied by progressive sterility, whereas wild-type F2 siblings displayed neither phenotype, but did possess discrete telomeric restriction fragments inherited from the mrt-1 background (Figure 1A, left panel, and data not shown). In addition, mrt-1 strains displayed late-onset chromosome fusions, as indicated by reduced numbers of metaphase-arrested meiotic chromosomes in late-generation mrt-1 mutants (Figure 1B and Supplementary Figure 1). The presence of end-to-end chromosome fusions was verified by isolation of X-autosome fusions from three independent mrt-1 strains (Figure 1C). Genetic mapping of the dominant-chromosome-loss phenotype of these independent fusions, which occurs when an X-autosome chromosome fusion is in trans to unfused chromosomes during meiosis, revealed tight genetic linkage of one end of the X chromosome with an end of an autosome in each case, confirming the formation of covalent end-to-end chromosome fusions (Figure 1C; see Supplementary data) (Ahmed and Hodgkin, 2000; Meier et al, 2006; Boerckel et al, 2007; Lowden et al, 2008). The former mrt-1 phenotypes resemble C. elegans strains that are deficient in telomerase activity in vivo (Ahmed and Hodgkin, 2000; Hofmann et al, 2002; Meier et al, 2006; Boerckel et al, 2007). Figure 1.Characterization and genetic mapping of mrt-1. (A) mrt-1 mutants display progressive telomere shortening. Southern blotting of genomic DNA with a telomere repeat-specific probe was performed as described previously (Ahmed and Hodgkin, 2000; Meier et al, 2006). F4 and F12 generations of three homozygous mrt-1(e2661) mutant and three homozygous wild-type siblings from a single outcross are shown in the left panel and three progressive generations of wild-type, mrt-1(yp2), two lines of mrt-1(e2661) and mrt-1(tm1354) each are shown in the right panel. Internal-repeat signals (Wicky et al, 1996) and telomere signals are indicated. (B) DAPI staining of late-generation wild-type or mrt-1 worms. Representative oocyte nuclei, indicated by dashed circles, are shown. (C) X-autosome fusions, eT3, eT7 and 9u, isolated from independent mrt-1(e2661) strains. Visible markers used for mapping are indicated. (D) Map position of mrt-1 as determined by three-factor crosses. The number of recombination events scored between mrt-1 and unc-29 is indicated in brackets. Cosmids covering the approximate genetic position of mrt-1 are shown. (E) mrt-1 gene structure and mutations. Point mutations are indicated in bold italics. Solid black boxes depict exons that are not translated due to the e2661 premature stop codon or are missing as a consequence of altered mrt-1 splicing in tm1354. In tm1354, the deletion within introns 3 and 5 leads to two alternatively spliced mRNAs indicated as (a) and (b) (see Supplementary Figure 2B), resulting in downstream exons to be out of frame (indicated in grey). (F) Western blot of protein extracts from wild-type and mrt-1 mutant strains. The arrow indicates MRT-1 protein, asterisks indicate nonspecific bands. Download figure Download PowerPoint Table 1. Progressive brood size reduction and loss of viability in late generation mrt-1, mrt-2 and trt-1 mutants Generation F2 F4 F6 F8 F10 F12 F14 F16 F18 F20 F22 F24 F26 Wild-type 1 W W W W W W W W W W W W W 2 W W W W W W W W W W W W W 3 W W W W W W W W W W W W W 4 W W W W W W W W W W W W W mrt-1(e2661) 1 W W W M M F VF S 2 W W W W W M M M F S 3 W W W W W M W M F VF S 4 W W W M W M M M F VF S mrt-1(yp2) 1 W W W W W M W M F F F VF S 2 W W W W W W W W M M M F VF 3 W W W W W W M M M M M M F mrt-1(tm1354) 1 W W W W W M W M F S 2 W W W W W M M F F F VF S mrt-2(e2663) 1 W W W W M M F M F VF VF S 2 W W M M M M M M F VF S 3 W W M W M M F F VF S 4 W W M F S trt-1(ok410) 1 W W W W M M M M F F VF S 2 W W W W W W M F VF S 3 W W W W W W W M M F F VF W, wild-type, ∼250 progeny per animal; M, medium, ∼80 progeny per animal; F, few, ∼20 progeny per animal; VF, very few, ∼3--5 progeny per animal; S, sterile. Mutants were backcrossed twice against wild-type to restore telomere length and two to four homozygous lines of the indicated genotype were followed by picking six L1s each line every two generations, as described previously (Ahmed and Hodgkin, 2000), for 26 generations. Brood size and sterility are indicated. Two- and three-factor crosses were used to map mrt-1 to approximately +2.92 on Chromosome I (Figure 1D). Although trt-1 is located nearby at +3.08, mrt-1 mutations complemented trt-1(ok410) for progressive sterility when propagated as trans-heterozygotes, whereas failure to complement was observed between the mrt-1 mutations e2661 and yp2, indicating that these mutations correspond to a single gene that is distinct from trt-1 (Table II; and data not shown). Failure to complement trt-1(ok410) was previously reported for three independent alleles of trt-1 (e2727, yp1 and tm899), thereby clearly defining the C. elegans telomerase reverse transcriptase (Meier et al, 2006). BLAST searches of predicted proteins to the left of trt-1 revealed an open reading frame, F39H2.5, encoding a protein with an N-terminal domain homologous to the second OB-fold of POT1 telomere-binding proteins and a C-terminal domain containing the metallo-β-lactamase and β-CASP motifs characteristic of the SNM1 family of nucleic acid processing factors (Figures 1E and 2, see below). Isolation and sequencing of the mrt-1 cDNA confirmed the predicted 608-amino-acid POT1 OB-fold/SNM1 dual-domain protein (Figure 1E and Supplementary Figure 2). Sequencing of F39H2.5 from wild-type, mrt-1(e2661) and mrt-1(yp2) revealed independent C-to-T transition mutations in e2661 and yp2, predicted to create stop codon and missense mutations, respectively (Figure 1E). The mrt-1(yp2) missense mutation results in an H127Y amino-acid change, thus altering an amino acid whose charge is conserved in the OB2-fold of most POT1 proteins (Figure 2A). Figure 2.MRT-1 shares sequence homology with POT1 and SNM1 proteins. (A) Protein domain structure of hPOT1, MRT-1 and the three additional C. elegans OB-fold domain proteins with homology to POT1. The conserved histidine H127Y (asterisk) mutated in the MRT-1 protein of mrt-1(yp2) is indicated. The region of homology around H127Y of the three C. elegans (C.e.) OB2-fold proteins aligned with the respective POT1 domains of Homo sapiens (H.s.), Xenopus laevis (X.l) and Saccharomyces pombe (S.p.) is shown. No clear alignment could be obtained for Arabidopsis thaliana POT1 within this region. Sequence alignments were generated using Pole BioInformatique Lyonnais ClustalW (http://pbil.ibcp.fr/htm/index.php). Red shading reflects sequence identity, green strong and blue weak similarity. (B) Protein domain structure of C.e. MRT-1, S. cerevisiae (S.c.) Pso2p and human SNM1A, SNM1B/Apollo and SNM1C/Artemis. A multiple sequence alignment of the HxHxDH and β-CASP motif-4 nuclease domains is shown below. Amino acids depicted on top of the alignment indicate amino-acid changes introduced into MRT-1 to generate MRT-1(4mut) (see Figure 3 and Supplementary data). Download figure Download PowerPoint Table 2. mrt-1 complementation Strain Lines Sterility trt-1(ok410) unc-29/++ 3 No* trt-1(ok410) unc-29/trt-1(ok410) unc-29 6 Yes mrt-1(e2661) dpy-5/++ 4 No* mrt-1(yp2) dpy-5/++ 3 No* ++trt-1(ok410) unc-29/dpy-5 mrt-1(e2661)++ 4 No* ++trt-1(ok410) unc-29/dpy-5 mrt-1(yp2)++ 4 No* +trt-1(ok410) unc-29/mrt-1(tm1354)++ 3 No* dpy-5 mrt-1(e2661)/+mrt-1(tm1354) 4 Yes dpy-5 mrt-1(yp2)/+mrt-1(tm1354) 4 Yes Trans-heterozygous analysis of mrt-1 alleles with trt-1. mrt-1 alleles were placed in trans to trt-1 or to a different mrt-1 allele (as shown) and progeny of several independent F1s were propagated as trans-heterozygotes (see Supplementary data) until sterility, while lines marked with an asterisk did not show any visible reduction in viability when propagated up to 15–20 generations. Upon identification of the mrt-1 gene based on our forward genetic experiments, a deletion of this locus, tm1354, was kindly generated by Shohei Mitani. The tm1354 deletion eliminates several exons of the C-terminal SNM1 nuclease domain, including conserved amino-acid motifs that are relevant for ICL repair in yeast Pso2p (Niegemann and Brendel, 1994; Li and Moses, 2003 and Figure 1E). RT–PCR of mrt-1 cDNA from tm1354 animals revealed two mRNAs predicted to result in truncated, out-of-frame proteins (Figure 1E and Supplementary Figure 2B). The tm1354 deletion was isolated under conditions that generate many additional lesions in a strain's genome (Gengyo-Ando and Mitani, 2000). Therefore, two- and three-factor crosses were performed to show that a locus tightly linked to the tm1354 deletion conferred progressive telomere erosion phenotypes characteristic of C. elegans telomerase mutants (Figure 1A and B and Table I, and data not shown). Further, the tm1354 deletion failed to complement the mrt-1 alleles e2661 and yp2 for progressive sterility and late-onset end-to-end chromosome fusion phenotypes, but complemented trt-1(ok410) (Table II). Although a strain containing the tm1354 deletion was previously mentioned to display progressive telomere shortening (Raices et al, 2008), the identification of independent alleles of this locus, as well as the genetic mapping and complementation tests described here, indicate that the telomere shortening observed in the tm1354 strain is caused by a defect in the mrt-1/F39H2.5 gene. A polyclonal antibody raised against full-length MRT-1 detected equivalent levels of full-length MRT-1 and MRT-1(H127Y) in wild-type and mrt-1(yp2) worm extracts, respectively (Figure 1F). Thus, the POT1-related OB2 domain of MRT-1 is required for telomerase activity in vivo. In contrast, no MRT-1 protein was detected in the e2661 nonsense mutation and tm1354 deletion extracts (Figure 1F and data not shown), indicating that these mutations are likely to be null alleles of mrt-1/F39H2.5, a non-essential gene required for de novo telomere repeat addition in C. elegans. mrt-1 encodes a dual-domain protein The N-terminal domain of C. elegans MRT-1 shares sequence homology with the second OB-fold of POT1 proteins (Figure 2A and Supplementary Figure 3). Single-stranded telomeric DNA-binding proteins commonly contain two adjacent N-terminal OB-folds, OB1 and OB2 (Horvath et al, 1998; Lei et al, 2003, 2004; Theobald and Wuttke, 2004). In addition to MRT-1, the C. elegans genome encodes two short proteins with homology to the second OB2-fold of POT1, F57C2.3 (CeOB1) and 3R5.1 (Figure 2A and Callebaut et al, 2002; Raices et al, 2008). The OB2-folds of MRT-1, F57C2.3 (CeOB1) and 3R5.1 are closely related and likely evolved from a single ancestral OB2-fold gene. A fourth C. elegans gene, B0280.10 (CeOB2), is homologous to the first OB-fold of POT1, OB1 (Figure 2A and Raices et al, 2008). The tandem OB-fold structure typical of POT1 proteins has been subjected to fission and duplication in C. elegans. Thus, we originally identified mrt-1/F39H2.5 based on the genetic map position of the mrt-1(e2661) telomerase-deficient mutant, and three additional C. elegans genes were identified based on their homology to POT1: B0280.10, F57C2.3 and 3R5.1. While this study was in progress, these genes were independently identified as POT1 homologues (Raices et al, 2008). We designate the C. elegans gene name for these genes as 'pot', for 'homologous to Protection of Telomeres 1(Pot1)', where pot-1 is B0280.10 (CeOB2) pot-2 is F57C2.3 (CeOB1) and pot-3 is 3R5.1 (Figure 2A) (Raices et al, 2008; Lowden et al, 2008). These genes display sequence similarity to, and evolved from, POT1, and their functions may reflect (1) one or more functions of ancestral POT1, including but not limited to 'protection of telomeres', or (2) derived functions that may be unrelated to the ancestral protein. Since F39H2.5/mrt-1 contains homology to two conserved proteins, POT1 and SNM1, the gene name mrt-1 is used, based on the Mortal Germline phenotype of mrt-1 mutants (Ahmed and Hodgkin, 2000). MRT-1 is the only C. elegans POT1-like OB-fold protein that is required for telomerase activity in vivo (Figure 1), whereas the pot-1(CeOB2) and pot-2(CeOB1) genes may repress telomerase activity or recombination at telomeres (Raices et al, 2008; M Lowden and S Ahmed, unpublished data). The C-terminus of MRT-1 corresponds to the sole C. elegans homologue of SNM1 proteins (Figure 1E and Figure 2B), which are members of the nucleolytic DNA- and RNA-processing β-CASP (metallo-β-lactamase-associated CPSF-Artemis-SNM1/PSO2) protein family (Aravind, 1999; Callebaut et al, 2002; Dominski, 2007). Saccharomyces cerevisiae Pso2p, and mammalian SNM1A and SNM1B/Apollo promote ICL repair (Henriques and Moustacchi, 1980; Demuth et al, 2004; Bae et al, 2008; Hazrati et al, 2008; Hemphill et al, 2008). In addition, SNM1B/Apollo and SNM1C/Artemis contribute to telomere end protection (Rooney et al, 2003; Freibaum and Counter, 2006; Lenain et al, 2006; van Overbeek and de Lange, 2006). The OB-fold/SNM1 dual-domain structure of MRT-1 is observed for the closely related Caenorhabditis species remanei and briggsae, but was not predicted from genome sequences of the distantly related parasitic nematodes Brugia malayi and Trichinella spiralis (data not shown). Thus, fusion of POT1 OB2 and SNM1 domains to create the mrt-1 gene may have occurred within the Nematode phylum. MRT-1 acts as a nuclease in vitro To determine whether MRT-1 harbours nucleolytic activity as implied by its sequence homology to SNM1 proteins, we purified wild-type, the MRT-1(H127Y) OB-fold mutant, and two putative nuclease-dead mutant versions of MRT-1, MRT-1(D245A) and MRT-1(4mut), from Escherichia coli (Supplementary Figure 4A). The MRT-1(D245A) protein contains an aspartate to alanine mutation in the conserved HxHxDH signature motif, which comprises residues predicted to participate in zinc coordination (histidines) and hydrolysis (aspartate) at the active site (Figure 2B and Carfi et al, 1995). In budding yeast, this mutation diminishes the in vitro 5′-to-3′ nuclease activity of Pso2p and leads to an ICL-repair defect in vivo (Li et al, 2005). The corresponding mutation abolishes the in vitro 5′ exonuclease activity of mammalian SNM1A (Hejna et al, 2007) and reduces the endonucleolytic activity of SNM1C/Artemis that is observed in the presence of DNA-PK (Pannicke et al, 2004). However, as this single amino-acid change does not abolish SNM1 nuclease activity in all cases (Pannicke et al, 2004), we also disrupted the putative catalytic core of MRT-1 with four mutations (4mut): a HxHxDH-to-AxAxAH triple mutation and a D335A substitution in motif-4 (Figure 2B and Poinsignon et al, 2004). Wild-type and mutant versions of recombinant MBP-6 × His–TEV–MRT-1 were purified over a TALON column resulting in a ∼120-kDa MBP-6 × His–TEV–MRT-1 band (Supplementary Figure 4A). Cleavage with TEV protease followed by a second purification step yielded untagged full-length MRT-1 (Supplementary Figure 4A and Supplementary data). Cleaved and purified MRT-1 protein exhibited 3′ nuclease activity as revealed by complete removal of one or more 3′ nucleotides from a 5′-end-labelled substrate (Figure 3A, arrow, and Figure 3B, arrow), accompanied by a smear of additional degradation products, which includes minor stalling points and release of the terminal 5′ nucleotide. This activity was not observed for TEV protease alone (Figure 3A, lane 11). Importantly, no nuclease activity was observed with MRT-1(D245A) or MRT-1(4mut) mutants, indicating that this activity requires conserved residues in the MRT-1 nuclease domain (Figure 3A, lanes 5–10; Figure 3B). A time-course experiment using MRT-1 and MRT-1 (H127Y), which contains the POT1 OB-fold substitution that abolishes telomerase activity in vivo, indicated that the nuclease activity of MRT-1, apparent by the complete removal of one or more 3′ nucleotides (arrow), as well as a smear of degradation products, is not affected by the OB-fold mutation (Figure 3B). Removal of the N-terminal epitope tags from MRT-1 promoted degradation of 5′-end-labelled substrates, accompanied by release of a small amount of 5′ mononucleotide, which might correspond to either a weak 5′ nuclease activity or a processive 3′ nuclease activity that completely degrades the oligonucleotide substrate (Figure 3A and B). To distinguish between these possibilities, MRT-1 was incubated with a 3′-end-labelled substrate. A single 3′ mononucleotide was released from 3′-end-labelled substrate in Figure 3C, in a nuclease domain-dependent manner. Thus, MRT-1 functions as a 3′–5′ nuclease, similar to the 3′–5′ exonuclease ExoI control (Figure 3C). If MRT-1 were a processive 5′-to-3′ nuclease, then a ladder of products would have been observed for the 3′-end-labelled substrate in Figure 3C. Consistently, incubation of various dilutions of MRT-1 protein with 3′-end-labelled G-strand substrate failed to reveal any cleavage intermediates expected for 5′-to-3′ exonuclease activity (Supplementary Figure 4E). We considered the possibility that the 5′ phosphate of 5′-end-labelled substrates might elicit 5′-to-3′ nuclease activity by MRT-1, as has been observed for SNM1 (Hejna et al, 2007), but addition of a cold 5′ phosphate to 3′-end-labelled ssDNA substrate did not promote the formation of an n-1 product by MRT-1, and resulted exclusively in release of the terminal 3′ nucleotide (data not shown). While MRT-1 and MRT-1(H127Y) showed comparable efficiency in cleaving the terminal 3′-labelled substrate, MRT-1(D245A) and MRT-1(4mut) exhibited strongly reduced kinetics and absence of activity, respectively (Figure 3C). Thus, our results indicate that MRT-1 functions as a 3′-to-5′ nuclease in vitro. Figure 3.MRT-1 acts as a 3′ nuclease in vitro. (A) 70 nM of MRT-1, MRT-1(D245A) and MRT-1(4mut) from different purification steps were incubated with 5 nM of a C. elegans telomeric G-strand oligonucleotide labelled at the 5′ end (asterisk). The line before the oligonucleotide sequence shown depicts an invariant linker sequence (see Supplementary data). 6 × His–TEV1 extracts were tested for contaminating nuclease activity (lane 11). The 5′-to-3′ nuclease RecJf and the 3′-to-5′ nuclease ExoI were used as controls (lanes 12 and 13, respectively). 1-nt products generated by RecJf and 2- to 4-nt products generated by ExoI are indicated. The arrow indicates the reduced size oligonucleotide band due to nuclease activity. For panel A, lane 2, it is difficult to assess the degree of nucleolytic activity for uncleaved MRT-1 due to a gel-running artefact. (B) Time-course experiment of MRT-1, MRT-1(H127Y), MRT-1(D245A) and MRT-1(4mut) nuclease activity on a 5′ labelled (asterisk) C. elegans telomeric G-strand oligonucleotide. 70 nM of protein were incubated with 5 nM of 5′ labelled oligonucleotide and aliquots were taken at the time points indicated. 2- to 4-nt products generated by ExoI and 1-nt products are indicated. The line drawn before the oligonucleotide sequenc