Supplemental Materials: Supplemental Results Search for Ver homologues by bioinformatics analyses To search for homologues of the Ver protein, 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 Proteins, 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 Protein Data Bank (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. 1 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 genes, 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 chromosome 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 chromosomes 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. 4 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.