Materials and Methods Drosophila Strains, Plasmids, and Constructs Drosophila stocks were maintained at 25u C on corn meal, molasses medium with dry yeast added to the surface. Oregon R was used as a control for all experiments. Full length mu2 mRNA was amplified from total RNA using RT-PCR and cloned into a pAGW destination vector. The C2 genomic fragment of mu2 that rescues the mutant phenotype [20] was cloned into pCaSpeR-DEST2 obtained from the Drosophila Genomics Resource Centre. The N- terminal (aa 1–250) and C-terminal domains (aa 1019–1259) of MU2-PB were cloned in frame at the EcoRI site into pGEX-4T1 to express them as GST fusion fragments. The expressed proteins were detected using mass spectrometry and were immobilized on glutathione sepharose (Amersham) for further studies. pCaSpeR- DEST2 was injected into y w1118 embryos, transformants were recovered, and the insertions were mapped genetically. Production of Anti-MU2 Antibodies A 59 fragment of mu2 (bp 37–678) was amplified from cDNA using primers containing the EcoRI restriction sites and cloned into pGEX4T1. The purified fragment was identified using mass spectrometry and was used to generate polyclonal antibodies in mice. Irradiation and Immunostaining of S2 Cells Exponentially growing S2 cells were seeded in 8 well chambered slides and transfected with pAGW-dMU2 plasmid using Effectene (Qiagen). Cells were exposed to 25 Gy of c irradiation using 137Cs at 5 Gy/min. Immunostaining of S2 cells was performed as described [43]. Cells were incubated with primary rabbit anti-cH2Av at 1:1000 dilution (Rockland Biochemicals, MD) and with mouse anti-MU2 antibodies (1:100). Slides were mounted in SlowFade Gold antifade reagent (Invitrogen) and were visualized using confocal microscopy. Figure 9. Model to explain new telomere formation in mu2 mutants. In a wild type genetic background a DSB induced by irradiation in a stage 14 egg chamber leads to the activation of a DNA damage response checkpoint and DNA repair. In mu2a mutants recognition of a DSB is impaired. Thus, the damage response checkpoint is not activated and DNA repair does not ensue. This leads to incorporation of the unrepaired break into the zygote. The lack of cell cycle control in the early embryo (until cycle 10) and the presence of relatively high levels of telomeric proteins (deposited maternally) may lead to the association of the DSB with these proteins and the establishment a new telomere. doi:10.1371/journal.pgen.1000473.g009 MU2 and Double Strand Break Repair MU2 RNAi in S2 Cells RNAi was performed in S2 cells according to established protocols [44,45]. Two regions of the mu2 gene, the N-terminal FHA domain, and the C-terminal tandem BRCT domains, were amplified from mu2 cDNA using primers with T7 promoter binding sites at the 59 and the 39 ends. As a negative control, we PCR amplified a 750 bp sequence from the bacterial cloning plasmid using the strategy described above. The PCR product was gel purified, an in vitro transcription reaction was performed, and the dsRNA was purified using Megascript T7 kit (Ambion Inc.) according to the manufacturers instructions. S2 cells were grown as described previously, plated at a density of 16105 and transfected with 1 mg dsRNA using Effectene (Qiagen) according to manufacturers instructions. Cells were exposed to 25 Gy of c irradiation using 137Cs at 5 Gy/min and immunostaining was performed as described in the previous section. Ovary Fixation and Immunofluorescence Fixation and staining of the ovaries was performed according to Scott and Hawley [46]. Ovaries were stained with mouse anti- MU2 serum (1:100), mouse anti-C(3)G antibodies (1:200), rabbit anti-Rad50 antibodies (1:200), or rabbit anti-cH2Av (1:1,000). The cH2Av foci were counted manually in the germaria that showed clear foci. Preparation of Nuclear Extracts Nuclear extracts were prepared from S2 cells using the protocol of Dingham et al. [47]. Protein concentrations were estimated and extracts were stored at 270uC. GST Pull-Down Assays A GST pull-down assay was performed to identify the region of MU2 that interacts with the MRN complex. Nuclear extracts were prepared from S2 cells as described above, were used as a bait and incubated with GST, GST-FHA, GST-BRCT fragments of MU2 that act as prey to capture the MRN group of proteins in the nuclear extracts. The GST preys were loaded onto the glutathione agarose beads and incubated with nuclear extracts prepared from S2 cells overnight in NETN (20 mM TrisHCl pH 8.0, 0.5 mM EDTA, 0.1% NP40, 100 mM NaCl) buffer containing 2% BSA. The beads were washed extensively to separate the unbound from the bound proteins, boiled in SDS sample buffer and analyzed by western blotting for the detection of proteins of MRN complex. Peptide Binding Synthetic peptides corresponding to the C-terminal tail of H2Av (N-KEETVQDPQRKGNVILSQAY-C) phosphorylated at the Ser residue were generated chemically, along with unphosphory- lated controls, and biotinylated at the N terminus (Sigma Genosys). The peptides were conjugated to streptavidin agarose beads. Pull-downs were performed using the nuclear extracts from S2 cells (Figure 5B) or the GST, GST-FHA, and GST-BRCT domains of MU2 expressed in bacteria (Figure 5A) acccording to Stucki et al. [36]. Bound proteins were subjected to immunoblot- ting to detect complexes from nuclear extracts and to SDS PAGE for the GST fragment pull-downs. Apoptosis and Determination of S-Phase Checkpoint Wing imaginal discs were dissected and stained with a the vital dye acridine orange as described previously [48,49]. Determina- tion of S phase checkpoint was performed according to Smolik and Jones [50] using Oregon R and mu2a flies. The total number of PLoS Genetics | www.plosgenetics.org 11 May 2009 | Volume 5 | Issue 5 | e1000473