Author Summary Telomeres are structures at the ends of eukaryotic chromosomes required for chromosome stability. If unrepaired, a single chromosome end without a telomere is sufficient to kill a cell, but new telomere formation is rare. Previously, we described a gene in Drosophila whose mutants, after irradiation, produced many progeny with chromosomes lacking a natural telomere. The new broken chromosome ends, however, bound telomeric proteins and behaved as telomeres. Here, we show that the protein encoded by this gene, a homolog of the human MDC1 gene, is a component of the repair foci that form at double strand DNA breaks and are prerequisite for both cell cycle arrest and DNA repair. The protein acts as a scaffold, connecting a phosphorylated histone that marks the site of the break to a protein complex necessary for repair. These results suggest a model for formation of neotelo- meres in which DNA breaks induced in mutant oocytes evade repair and are deposited into embryos, which contain an abundance of maternally deposited telomeric proteins. In this context a chromosome end not recog- nized as broken may be treated as a telomere. These results may provide a basis to understand neotelomere formation. males are mated [19], these lesions do not potentiate terminal deficiencies when the female carries the mu2a mutation, suggesting that the DNA repair machinery, per se, is not affected by this mutation. In addition, the transmission of DSBs from the oocyte to the zygote suggests a defect in cell cycle regulation. We have endeavored to identify the function of MU2 and understand phenotype of mu2a at the molecular level. mu2 mRNA encodes a polypeptide of ,139 kDa [20] that has a tandem BRCA1 C-terminal (BRCT) domain at the C terminus. We show here that the MU2 protein is associated with ionizing radiation- induced foci in somatic cells and recombination foci during meiosis. The N terminus of MU2 contains a forkhead associated (FHA) domain that interacts with the MRN complex, while the BRCT domain interacts with histone variant H2Av phosphory- lated at Ser 137 (cH2Av). This histone variant is homologous to mammalian H2AX [21], which when phosporylated marks DNA repair foci. MU2 also plays a role in the regulation of the intra-S checkpoint in eye-antennal imaginal discs. Based on amino acid alignment, functional similarities, domain architecture, and protein-protein associations, we propose that mu2 is an ortholog of human mediator of DNA damage checkpoint 1 (MDC1) gene. Sequencing of the mu2a cDNA revealed a frameshift mutation at aa 1065 leading to a stop codon and loss of the BRCT domain. The delay in the repair of radiation induced lesions in conjunction with the loss of cell cycle control in mu2a mutants suggests a novel role of the mutation in the formation of new telomeres at the site of a DSB. Results Location of MU2 In Vivo Genetic studies suggested that oocytes mutant for mu2 have a defect in the processing of DSBs induced by ionizing radiation and produce terminally deficient chromosomes at a high frequency [12,17]. Based on phenotypic differences between males and females, it was proposed that the MU2 protein is a component of the oocyte nucleus. To understand the localization of MU2, transgenic animals expressing modified GFP (mGFP) tagged MU2 MU2 and Double Strand Break Repair were made. Oocytes from these females showed that MU2 is concentrated in the oocyte nucleus at several stages of develop- ment (Figure 1A), although the cytoplasm of more advanced oocytes was also fluorescent. The testes of transgenic males do not show any MU2 localization to the germ cells (Figure 1B), although tagged MU2 localized to the somatic sheath cells, as was found for mu2 mRNA [20]. Further, the distribution of MU2 in larval somatic tissues appeared to be uniform at low magnification over the imaginal discs (Figure S1). In salivary glands MU2 was concentrated on polytene chromosomes with a distribution similar to that of the DAPI stain (Figure 1C). To study the function of the unmodified MU2 protein, we generated antibodies to an N-terminal fragment of MU2. Antibody staining of Oregon R ovaries confirmed that MU2 was concentrat- ed in the oocyte nucleus at several developmental stages (Figure 1D). A ring shaped localization of MU2 protein was also observed in the cytoplasm of more mature developing egg chambers (Figure S2). In contrast, mu2a mutants showed low levels of MU2 in their ovaries and poor nuclear localization (Figure 1E). Analysis of the mRNA levels of mu2 using RT PCR showed no significant difference between the wild type and in the mutants, suggesting a lack of nonsense mediated decay. Western analysis of larval and the adult tissues, however, showed a decrease in levels of MU2 protein. Further, the mutants showed a single band for MU2, rather than the two bands seen in wild type (Figure 1F). The presence of a single band with a decreased intensity is consistent with the low signal that we see in immunofluorescence studies. Sequencing of the cDNA from the mu2a mutant showed that the C-terminal BRCT domain of the protein is obliterated due to a frameshift mutation at amino acid 1065 that leads to a premature stop codon. It is possible that the defective protein is rapidly cleared. In Drosophila females meiosis occurs within a 16 cell cyst in the germarium at the anterior end of the ovary. The cyst initially contains two pro-oocytes and 14 nurse cells. The two pro- oocytes enter meiosis simultaneously and generate DSBs, although only the actual oocyte nucleus enters the pachytene stage. The other pro-oocyte becomes a nurse cell [22,23]. We used the antibody to MU2 to examine localization in the germarium. Most of the staining for MU2 was in regions 2A and 2B, which stain specifically with cH2Av antibody. MU2 co- localized with this DSB marker (Figure 2A). However, the germaria of mu2a females showed very little to no staining with MU2 antibody (Figure 2B). mu2a germaria also showed a decrease in the number and the intensity of cH2Av foci when compared to wild type (Figure S3), suggesting that the presence of MU2 affects the formation or stability of the cH2Av foci. The decrease in the number of cH2Av foci (Figure 2C) is similar to the decrease in the meiotic recombination seen in the mutant [18]. In both control and mutant oocytes the DSBs were resolved by region 3, as shown by the lack of staining by cH2Av antibody (Figure 2A). MU2 Forms Foci in S2 Cells One of the earliest known responses to DSB formation is the phosphorylation of the C- terminal tail of the variant histone H2A located in the vicinity of the DNA breaks [24]. Antisera to mammalian phosphorylated H2AX (cH2AX) recognize Drosophila cH2Av, suggesting a strong homology [21]. cH2AX binds proteins such as MDC1 (through the latter’s C-terminal BRCT domain) and acts as a platform for the assembly of DDRs into repair foci. MU2 has a C-terminal tandem BRCT domain, which might interact with cH2Av. We tested this prediction initially by transfecting S2 cells with eGFP-tagged MU2. Unirradiated cells showed a broad distribution of MU2 protein over the nucleus and PLoS Genetics | www.plosgenetics.org 2 May 2009 | Volume 5 | Issue 5 | e1000473