Supplemental Materials:

Supplemental Results

Search for Ver homologues by bioinformatics analyses To search for homologues of the Ver , we used the procedure outlined in Supplemental Fig. S2. We first searched by CSI-BLAST (Biegert and Soding 2009) non- redundant (NR) protein sequence database (including all non-redundant GenBank CDS translations, RefSeq , PDB, SwissProt, PIR and PRF) using the full-length amino acid sequence of Ver as query (UniProtKB entry code: Q9VTZ2). Two iterations of CSI-BLAST searches yielded 22 unique sequences, with an Expectation (E) value threshold of 10-5. These sequences included 11 proteins from different Drosophila species; 5 OB-fold containing proteins (OBFC1) from Homo sapiens, Bos taurus, Rattus norvegicus, Mus musculus and Xenopus tropicalis, all sharing homology with yeast Stn1 (Miyake et al. 2009; Wan et al. 2009); and 6 proteins annotated as “unnamed” (from EMBL submitted sequences) or “predicted” (automatically provided to the protein database from GeneBank sequence data). We discarded unnamed/predicted proteins due to possible mistakes in their annotation. The remaining sequences were retrieved and aligned with PROMALS3D (Pei et al. 2008) with default parameters (Supplemental Fig. S3A). We then generated a hidden Markov model (HMM) based on the multiple sequence alignment (MSA) of Ver orthologous found in the 12 sequenced Drosophila species (Clark et al. 2007; Supplemental Fig. S3B). Using this HMM and the HHpred server (Soding et al. 2005) we searched the Conserved Domain Database (CDD) at the NCBI. The highest scoring hit was the hOBFC1_like subfamily of OB-fold (OBF) domains (CDD ID: cd04483, E-value of 1.6x10-5; probability score of 97.50%), which belongs to the Rpa2 OB-fold (OBF) family. The OBF domains of the hOBFC1_like subfamily are found in the human and mouse homologues of yeast Stn1 (Miyake et al. 2009; Wan et al. 2009). The HMM and the HHpred server were also used to search a collection of HMM profiles derived from proteins included in the (PDB). The highest scoring hit was the OBF domain of S. pombe Stn1 (PDB ID, 3KF6 chain A; Sun et al. 2009), with an E-value of 8.2x10-13 and a probability score of 99.42%. The second and third hits were the OBF domains of human RPA2 (PDB ID, 2PI2 chain A; E-value, 2x10-7; probability score, 98.49%; Deng et al. 2007) and Candida tropicalis Stn1 (PDB ID, 3KF8 chain A; E-value, 3.07; probability score, 88.03%; Sun et al. 2009), respectively.

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Furthermore, we searched for Ver homologues using several fold recognition methods (FFAS03, mgenthreader, Phyre, FUGUE, SAM-T08) in the GeneSilico metaserver (Kurowski and Bujnicki 2003). With all these methods, the highest scoring hit was the OBF domain of S. pombe Stn1 (Supplemental Fig. S2 and Fig. 4A). Collectively, our searches indicate that OBF domain of fission yeast Stn1 is the closest homologue of the Ver OBF. Thus, based on the GeneSilico alignments, and using the S. pombe Stn1structure as template (Sun et al. 2009), we built a model of the Ver core portion (residues ~ 37 to 205) using the “FRankestein’s monster” approach (Kosinski et al. 2003; Supplemental Fig. S2 and 4B). Our model of Ver has been evaluated as a potentially ‘very good model’ by the PROQ model quality assessment program (Wallner and Elofsson 2006) and it is predicted to have a root mean square deviation of ~ 3 Å to the (currently unknown) native structure according to MetaMQAP server (Pawlowski et al. 2008). In summary, the Ver three-dimensional model is likely to be sufficiently accurate for making functional inferences. We next compared the predicted Ver structure (Fig. 4B) with the protein structures in the PDB database using the DALI program (Holm et al. 2008). We found significant similarities with several OBF containing proteins. These proteins included human RPA70 (hRPA70), whose structure has been determined in complex with the cognate ssDNA (Bochkarev et al. 1997). Thus, we superimposed the Ver model to the hRPA70 structure to generate a model of the Ver OBF domain bound to ssDNA (Fig. 4C). Despite the low overall sequence identity, the positions of some amino acids in the DNA-binding region of hRPA70 are similar to those of the corresponding residues in the Ver model. The F89 and F169/R170 (F169 and R170 are next to each other) residues of Ver superimpose reasonably well with the K343 and F386 residues of hRPA70 (Fig. 4C), which are responsible for protein/DNA interactions (Bochkarev et al. 1997). The Ver/DNA model also suggests that K91 might be involved in protein-DNA interactions because it protrudes from the face of the OB-fold domain predicted to bind DNA (Fig. 4C).

Loss of Ver does not trigger the spindle checkpoint (SAC) response We have recently shown that HOAP-depleted telomeres recruit the BubR1 kinase and trigger the spindle assembly checkpoint (SAC) response, causing a metaphase arrest phenotype (Musarò et al. 2008). We thus asked whether Ver-depleted telomeres cause the SAC response. ver2 mutants showed an anaphase frequency of 12,0% (n = 150), substantially higher than that observed in cav mutants (1,8%; n = 220), and similar to wild type (13,3%; n=150). In addition, the frequency of BubR1-labeled telomeres, not involved in telomeric 2 fusions, was 6% (n = 150 telomeres) in ver2 mutants, and 25% (n = 100 telomeres) in cav mutants. These results are consistent with the observation that moi mutants do not trigger the SAC response (Raffa et al. 2009); they support the notion that the SAC response is specifically induced by telomeric accumulations of BubR1 (Ciapponi and Cenci 2008; Musarò et al. 2008).

Divergence of telomere-capping proteins in Drosophila species Consistent with the findings that cav, moi and ver are fast-evolving , a simple pairwise comparison in amino acid sequence revealed that HOAP, Moi and Ver display a higher divergence in sequence identity than most Drosophila proteins required for telomere protection (Supplemental Table S1). The only non-terminin protein with a relatively high sequence divergence is Nbs (Supplemental Table S1). However, Nbs is the most variable subunit of the conserved MRN complex (D'Amours and Jackson 2002) and its divergence among species might not be related with its function at telomeres. Collectively, our results indicate that the terminin components have evolved more rapidly than the other Drosophila proteins that prevent telomere fusion.

Supplemental Materials and Methods

Drosophila Strains The ver1 allele was isolated from a collection of 1680 EMS induced third late lethals, generated in Charles Zuker’s laboratory. A cytological screen of larval brain squashes from these lethal lines yielded 16 mutants with frequent telomere-telomere attachments. Complementation tests showed that these 16 mutants identify 9 genes, one of which was ver1. The ver1 allele was characterized by DNA sequence analysis; genomic DNA isolated from either mutant or control larvae from the isogenic stock used for EMS mutagenesis was amplified by PCR and sequenced with an automatic DNA sequencer. All ver mutations and Df(3L)sex204 (a deficiency that removes part of the the genomic region that contains ver) were balanced over TM6B or TM6C, which carry the dominant larval marker Tubby. Homozygous and hemizygous mutant larvae were recognized for their non-Tubby phenotype. All stocks were maintained on standard Drosophila medium at 25 °C.

Chromosome cytology and immunostaining DAPI-stained larval brain chromosome preparations were made as described (Cenci et 3 al. 1997). To obtain metaphase preparations for immunostaining, brains were dissected in 0.7% sodium chloride, incubated for 45 min with 10-5 M colchicine in 0.7% sodium chloride, treated with hypotonic solution (0.5% sodium citrate) for 7 min, and fixed according to (Cenci et al. 2003). Polytene chromosome preparations were obtained as described (Siriaco et al. 2002). For double immunostaining of HOAP and HP1, polytene were incubated overnight with both the anti-HP1 C1A9 mouse monoclonal (James et al. 1989) and an anti-HOAP rabbit polyclonal (Badugu et al. 2003) antibody, diluted 1:50 and 1:200 in PBS, respectively. The primary antibodies were detected by simultaneous incubation for 2h at room temperature with fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse IgG (1:200) and CyTM3- conjugated donkey anti-rabbit IgG (1:200), both from Jackson Laboratories. Mitotic and polytene chromosome preparations were analyzed using a Zeiss Axioplan epifluorescence microscope equipped with a CoolSnap ES camera (Photometrics) as described (Cenci et al. 2003). For in vivo detection of Ver-GFP, GFP-Moi and HOAP-GFP, salivary glands were dissected in Voltalef oil and immediately analyzed under a Zeiss Axiovert 20 fluorescence microscope. Images were acquired with a CoolSnap HQ camera (Photometrics) using a 2x2 bin. Image acquisition was controlled through a Metamorph software package (Universal imaging, Downing Town, Pennsylvania). Eight fluorescence optical sections were captured at 1-µm z steps; each fluorescent image shown is the maximum-intensity projection of all sections.

RT-PCR Total RNA from either wild type or mutant larvae was purified using the Qiagen RNeasy kit. cDNAs were synthesized using the SuperScript III system (Invitrogen) and used as templates in PCR reactions to amplify either the ver or the RpL12 (control) transcript. A 645-bp fragment identifying the entire ver coding sequence (CDS) was amplified using the (5’-CACCATGGATTTTAATCAGAGTTTCGAGGACATAG-3’) and (5’-TTTATTTGTTGTATTCTGCATTG-3’) primers. The RpL12 transcript was amplified using the (5’-CTTTCTTTTTCTGTGTGT-3’) and (5’-GGGTCAATTATTACAGACTG-3’) primers. The RNAs of all ver-GFP transgenes were amplified using the (5’-CACCATGGATTTTAATCAGAGTTTCGAGGACATAG-3’) and (5’ –GGCATGGACGAGCTGTACAAG- 3’) primers.

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Complementation analysis Complementation analysis between ver+-GFP transgenes and ver mutations was carried out using two homozygous viable lines, each carrying the transgene on the second chromosome. ver+-GFP /CyO; ver/TM6B flies were mated inter se and the ver+-GFP/CyO; ver/ver offspring was examined for viability and the presence of telomere fusions. Two UAS-cav+-GFP transgenic lines, both with the transgene on the second chromosome were used in complementation tests with cav mutations. We generated by recombination chromosomes bearing both UAS-cav+-GFP and the actGAL4 driver. We then constructed UAS-cav+-GFP actGAL4/CyO; cav1/TM6B flies that were mated inter se. Examination of the UAS-cav+-GFP actGAL4/CyO; cav/cav progeny revealed that both transgenes used for complementation rescue both the lethality and the telomere phenotype of cav mutants.

Cell transfection S2 cell cultures were transfected with both cav-FLAG and moi-HA plasmids using Cellfectin (Invitrogen) following the manufacturer’s instructions. Seventy-two hours after transfection, cells were harvested and extracts were prepared by lysing cells in 150 mM NaCl,

50 mM Tris-Hcl pH 7.5, 30 mM NaF, 25 mM β-glycerophosphate, 0.2 mM Na3VO4, Triton X-100 1%, and Complete protease inhibitor mixture tablets (Roche).

Protein purification Recombinant His-Ver Proteins were expressed in strain BL21 (DE3). Exponentially growing cells were induced at 37 °C for 4h by the addition of 2 mM IPTG. Cells were harvested, resuspended and incubated for 1h in lysis buffer (400 mM NaCl, 100 mM KCl, 10% glycerol, 0.5% TritonX-100, 10 mM Imidazole, 50 mM Phosphate buffer pH 7.8, 0.2% lysozime, Complete protease inhibitors Roche). Lysates were then sonicated for 20 sec, incubated for 30 min with 1.5% N-Lauryl Sarcosyn, and centrifuged for 25 min at 4 °C. The soluble portion was then incubated 1 h with the Ni-NTA His-Bind Resin (Novagen), extensively washed with lysis buffer containing 20 mM imidazole. His-Proteins were then eluted with lysis buffer containing 250 mM imidazole.

Calculation of dN values ver, moi and cav CDSs were retrieved from FlyBase. The orthologues of these genes in the 11 sequenced Drosophila species (Clark et al. 2007) were identified by homology searches. Nucleotide sequences were aligned using the T-coffee software (Notredame et al. 5

2000) (default parameters) and the MSA was employed for calculation of non-synonymous substitutions per non-synonymous site (dN). All pairwise comparisons and between ver, moi, cav, Su(var)205 and RpL12 genes from D. melanogaster and their homologues from other species were carried out using the MEGA4 software (Kumar et al. 2008).

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Supplemental Figures

Supplemental Figure S1. Expression of ver and ver-GFP in different genetic backgrounds. (A) ver mRNA levels in larvae of the indicated genotypes. Note that the ver is normally transcribed in both ver1/ver1 and wild type (Or-R) larvae, whereas ver2 mutant larvae do not show detectable levels of ver mRNA. RpL12, a ribosomal protein-coding gene, was used as control (Ctr). (B) Transcription of ver-GFP transgenes in larvae of the indicated genotypes. (C, D) Immunoblotting of mixed larval brain and salivary gland extracts expressing ver+-GFP transgenes (C, D) or mutant ver1-GFP transgenes (C) in different genetic backgrounds (Or-R; moi or cav mutant). Note that Ver+-GFP is expressed in all genetic backgrounds, while Ver1-GFP is not detectable (C). Ver-GFP proteins were detected with an anti-GFP antibody; asterisk, unspecific band; L.C., loading control.

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Supplemental Figure S2. A flow chart describing the bioinformatic strategies used to search for Ver homologues and to model the Ver structure (see text for details). CSI-BLAST: context-specific version of standard NCBI PSI-BLAST; MSA: Multiple Sequence Alignment; HMM: hidden Markov model: HHpred: web server for protein remote homologs searching and three dimensional protein structure prediction; CDD: Conserved Domain Database, NCBI; PDB: Protein Data Bank; NR database: non-redundant protein sequence database, NCBI; GeneSilico metaserver: gateway to various methods for protein structure prediction and analysis; FRankentstein’s monster method (FR, fold recognition): a comparative modeling approach that merges the finest fragments of Fold-Recognition models using iterative model refinement aided by 3D structure evaluation.

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B

Supplemental Figure S3. Ver is an Stn1-related protein. (A) Multiple sequence alignment of Ver homologues obtained by CSI-BLAST on non-redundant protein sequence database. The top line (Ver_SS_consensus) shows the predicted secondary structure (residues in β-sheets and a α-helices are marked with E and H, respectively). (B) Multiple sequence alignment of 12 Drosophila orthologous Ver proteins. The top line shows the predicted secondary structure as green cylinders (α helices) and red arrows (β strands). The color saturation of the shaded columns indicates the conservation level of each position in the alignment. 9

Supplemental Figure S4. Functional analysis of the Verm4-GFP mutant protein. (A) In vivo localization of Verm4-GFP in salivary gland nuclei, showing that the mutant protein localizes at the telomeres. (B) ver mutant larval neuroblasts expressing the Ver+- GFP or the Verm4-GFP protein. While Ver+-GFP rescues the telomere fusion phenotype, expression of Verm4-GFP does not suppress end-to-end fusion.

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Supplemental Figure S5. Ver is not required for HOAP and HP1 localization at telomeres. (A) Mitotic chromosomes from ver2 mutants immunostained for HOAP (red) and counterstained with DAPI. HOAP localizes at both free and fused (arrows) telomeres. In Oregon R controls, 91% of telomeres (n = 280) displayed a clear HOAP signal. Similarly, in ver2 mutants, 86% of the telomeres not involved in fusion events (n = 110) and 60% of the attached telomeres (n = 150) showed a HOAP signal. (B) Wild-type (Or-R) and ver2 polytene chromosomes immunostained for both HOAP (red) and HP1 (yellow) and counterstained with DAPI. Note that mutant telomeres accumulate normal amounts of HOAP and HP1.

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Supplemental Figure S6. Ver and Moi are HOAP- and mutually-dependent for their localization at polytene telomeres. In vivo localization of HOAP-GFP, GFP-Moi and Ver- GFP in salivary gland nuclei from larvae expressing the corresponding transgenes in either a cav, moi or ver mutant background. Note that HOAP-GFP localizes normally in both moi and ver mutant salivary gland nuclei. In contrast, Ver-GFP does not accumulate at polytene chromosome telomeres of cav and moi mutants. Similarly Moi-GFP does not accumulate at the telomeres of cav and ver mutants.

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Supplemental Figure S7. The terminin components are encoded by fast-evolving genes. The mean numbers of non-synonymous substitutions per non-synonymous site (dN values, represented as bars ±SD) for ver, cav and moi increase dramatically with the increase of the evolutionary distance between D. melanogaster and each of the 11 Drosophila species used for comparison. The Su(var)205 gene encoding the non-terminin telomere- capping protein HP1 exhibits relatively modest increases in dN values, while the RpL12 gene shows very little variation in dN values. dN values have been calculated using the KUMAR (Kumar 2000), LWL85 (Li et al. 1985) and PBL93 (Pamilo and Bianchi 1993) methods. D. sim, D. simulans; D. sec, D. sechellia; D. yak, D. yakuba; D. ere, D. erecta; D. ana, D. ananassae; D. pse, D. pseudoobscura; D. per, D. persimilis; D. wil, D. willistoni; D. moj, D. mojavensis; D. vir, D. virilis; D. gri, D. grimshawi.

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Supplemental Table S1. Identity percentages (id%) between telomeric proteins from D. melanogaster and their homologues from 11 sequenced Drosophila species (http://flybase.org/blast/; Clark et al. 2007). The id% is the percentage of identical matches between the two amino acid sequences calculated by the EMBOSS Pairwise Alignment Algorithms (Mackey et al. 2002; http://www.ebi.ac.uk/Tools/emboss/align/index.html) using default settings.

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Supplemental References

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