very little cH2Av (Figure 3A). However, after irradiation, MU2 re- localized to ionizing radiation induced foci (IRIF) and co-localized with cH2Av (Figure 3A). Similar results were obtained with anti-MU2 antibodies. Staining S2 cells with an anti-MU2 antibody showed that MU2 was distributed broadly in the absence of radiation (Figure 3B). After irradiation, distinct foci of MU2 were observed that were nuclear in nature and co-localized with cH2Av foci (Figure 3B). We further studied the status of the foci at different times after irradiation. MU2 foci appear immediately after irradiation concomitantly with the appearance cH2Av foci (Figure 3C), suggesting that MU2 is intricately involved with recognition of DSBs and the formation of repair foci. To further understand the role of MU2 in the formation of cH2Av foci, we treated S2 cells with dsRNA, targeting two different regions of mu2. RT PCR analysis after 4 days of continuous treatment showed a decrease in the levels of mu2 RNA. S2 cells treated with either N-terminal or C-terminal dsRNA were used to study the effects on cH2Av foci formation. Cells were irradiated, fixed at two different intervals and stained with rabbit anti-cH2Av antibodies to detect cH2Av foci. As shown in Figure 4, a decrease in the levels of mu2 caused a decrease in the number of cH2Av foci immediately after irradiation. However, fixing the cells 15 minutes after irradiation showed an increase in the number and the intensity of foci, although these parameters did not reach wild type levels. These results suggest that MU2 affects the kinetics of foci formation. Physical Interaction of MU2 and Phosphorylated H2Av To further understand the nature of the interaction between MU2 and cH2Av, we performed a peptide pull-down assay using the C-terminal amino acids of H2Av phosphorylated at the conserved serine at position 137 as a probe for the bacterially expressed N- (aa 1–250) and C-terminal (aa 1019–1259, BRCT) domains of MU2 (Figure 5A). The BRCT domain, indicated by an asterisk, interacted specifically with the phosphorylated H2Av peptide. The N terminus of MU2, however, did not show any interaction with either the phosphorylated or the unphosphory- lated peptides. The bands that are observed in the lanes of unphosphorylated peptide at the positions of the GST inputs were confirmed to be of bacterial origin by mass spectrometry, suggesting that the unphosphorylated peptide does not show any binding to the MU2 domains in vitro. The direct physical interaction between the BRCT domain of MU2 with the phospho- peptide suggests that MU2 is an adaptor protein, and that the N terminus might act as a base for the recruitment of other proteins. Using a peptide pull-down assay, we analyzed the proteins that may form a complex with MU2 in nuclear extracts of S2 cells. Immunoblots were performed on the pull-down lanes using antibodies to the components of the MRN complex. MRE11, RAD50 and NBS were detected in the cH2Av lane of the pull- downs (Figure 5B). These results suggest that while binding to the cH2Av at the C terminus, MU2 also binds a complex of proteins, possibly at the N terminus, and thereby recruits the DDR complex to the site of a DSB. The MU2 FHA Domain Interacts with the MRN Complex Domain searches identified the presence of an FHA domain in the N-terminal region of D. melanogaster MU2 (aa 9–106) and five other Drosophila species [25,26]. Proteins containing FHA domains bind phosphorylated serine and threonine residues of proteins involved in DNA damage signaling. Since MU2 is a component of MU2 and Double Strand Break Repair meiotic recombination foci and radiation induced foci, we set out to identify the domain of MU2 that interacts with MRN complex. In the vitellarium of the Drosophila ovary, RAD50 is found in somatic follicle cells and oocyte nuclei (Figure 6A). Staining of the germarium with anti-RAD50 antibody showed that MU2 and RAD50 colocalize at the sites of recombination foci (Figure 6B). These results suggest the presence of a multiprotein complex, including cH2Av, MU2 and the MRN complex, that is involved in the recognition and repair of a DSB. To identify the domain of MU2 that interacts with the MRN complex, we created GST fragments of the N-terminal FHA and the C-terminal BRCT domains of MU2 and incubated these fragments with nuclear extracts from S2 cells. Immunoblot analysis of GST pull-downs using antibodies to MRE11, RAD50 or NBS showed that the N- terminal FHA domain of MU2 interacts with the MRN complex, confirming the presence of a functional FHA domain at the N terminus (Figure 6C). We next asked whether there could be a genetic interaction between MU2 and the components of the MRN complex. Mutations in the genes of the MRN complex are homozygous pupal lethal, and the wing imaginal discs of these homozygous animals show high levels of spontaneous apoptosis. Mutations in certain components of the DDR pathway are known to decrease the levels of spontaneous apoptosis. Thus, we predicted that, if MU2 is a transducer of the signal to repair a spontaneous or an induced DSB, mu2a should reduce the number of apoptotic cells. We examined apoptosis in wild type, homozygous single and double mutants of nbs1 and mu2a. mu2a mutants do not show any increase in apoptosis compared with the controls, whereas the nbs1 mutants show high levels of spontaneous apoptosis (Figure 6D). Interestingly, there is a considerable decrease in the levels of spontaneous apoptosis in the wing discs of the mu2a nbs1 double mutants (Figure 6D). However, mu2a did not significantly decrease the levels of apoptosis in the rad50 and the mre11 mutants. These results suggest a possible role for NBS separate from the MRN complex in apoptotic signaling and repair, as has been suggested previously [27,28]. MU2 Is Homologous to Human MDC1 If MU2 plays a conserved role in the formation of IRIFs, homologs in other organisms should be evident. A blast search of insects outside of diptera shows only one strong hit (E = 6.8e–9), which is to a protein ‘similar to mediator of DNA damage checkpoint 1’ (MDC1) in the wasp, Nasonia. Similarity extends along the carboxy half of the Drosophila MU2 protein from aa 581 to 1251. A blast of the human genome reveals only one hit, to MDC1 (E = 6e–6). The similarity, however, is restricted to the BRCT domain. Alignments of Drosophila MU2 isoform PB and human MDC1 using blast2 indicate more extensive homology to MDC1 (aa 289–1203 of MU2). Thus, the only region that does not show similarity between Drosophila MU2 and human MDC1 is the N terminus, which carries an FHA domain in both proteins. As in MDC1, MU2 contains several threonine-glutamine (TQ) dipeptides in the N-terminal half of the protein (Figure 7). MU2 Is Important for the Intra-S Checkpoint If MU2 is a homolog of MDC1, it is possible that it modulates the intra-S checkpoint [29]. As eye imaginal discs develop, a morphogenetic furrow passes over the disc from posterior to anterior. In this furrow cells undergo a final wave of synchronous mitoses. Thus, the third instar eye disc displays a row of mitotic cells. To test whether a mutation in mu2 has an effect on cell cycle control, eye discs were treated with 0.05 M hydroxyurea (HU) for 2.5 h, and mitotic cells were detected by antibodies to histone H3 phosphor- PLoS Genetics | www.plosgenetics.org 4 May 2009 | Volume 5 | Issue 5 | e1000473