MU2 and Double Strand Break Repair Figure 5. Physical interaction of MU2 and H2Av. Peptide pull-down assays were performed using chemically synthesized peptides of H2Av (phosphorylated and unphosporylated) to examine the interaction between bacterially expressed domains of MU2 and the peptides of H2Av. The H2Av peptides were biotinylated at the N terminus, which was used to conjugate them to the streptavidin agarose beads. These beads were then incubated with bacterially expressed and purified GST, GST-FHA, GST-BRCT domains of MU2. Panel (A) shows a coomassie-stained SDS polyacrylamide gel of an H2Av peptide pull-down experiment (cH2Av indicates the phosphopeptide; H2Av indicates the unphosphorylated peptide). Expressed GST-FHA and GST-BRCT domains, and bands appearing at the same molecular weight in other lanes, were identified using mass spectrometry. The GST-BRCT domain is pulled down specifically by the phosphorylated form of the peptide (position of the band indicated by an asterisk). (B) Biotinylated H2Av (phosporylated or unphosphorylated) peptides were incubated with nuclear extracts from S2 cells to isolate the MRN complex. Ten mg of total protein from the nuclear extract was loaded onto agarose beads. At the end of incubation the beads were washed, and the MRN complex was detected by Western analysis. doi:10.1371/journal.pgen.1000473.g005 and oocyte pronuclei after fertilization suggested that MU2 might be chromosomal or at least restricted to the oocyte nucleus. Chromatin structure is known to play a key role in the repair of DSBs, such that the sites of DSB are devoid of conventional nucleosomes [34] and many key chromatin components, such as HP1 [35]. Our studies show that the MU2 protein is nuclear and at least in salivary cells localizes to chromosomes, confirming its association with chromatin. Further, the presence of MU2 is important for the formation and/or the stability of IRIFs, which in turn are necessary for alterations in chromatin structure that are prerequisite for repair. MU2 Is an Ortholog of Human MDC1 We propose an explanation for the mu2a mutant phenotype by suggesting that mu2 is an ortholog of human MDC1. MDC1 forms a major component of IRIFs in mammalian cells by binding cH2AX using its C-terminal BRCT domain [36] and the MRN complex using its N-terminal FHA domain. Thus, MDC1 acts as a scaffold for the assembly of the IRIF, the multiprotein complex in which DSB repair occurs. MEFs isolated from MDC1 mice do form cH2AX foci, but the kinetics of focus formation are affected. We used RNAi of MU2 in S2 cells to understand the role of MU2 in the formation of cH2Av foci. Our studies indicated that foci are formed after irradiation, but their number and intensity are reduced. Further, the kinetics of formation are affected. These results are in agreement with a decrease observed in the germarium of the ovaries. MDC1 plays an important role in the regulation of the intra-S phase [37] and G2/M checkpoints [38] by regulating the corresponding effecter proteins. It also enhances NHEJ at dysfunctional telomeres [39], showing that proteins involved in recognition of DNA damage are important in telomere dynamics. MDC1 knockout mice are viable, although male MDC12/2 mice are sterile, suggesting a defect in spermatogenesis or meiosis [40]. MDC1 knockout mice and the cell cultures established from them, however, are hypersensitive to ionizing radiation. The phenotypes of the MDC12/2 mice were very similar to those observed for H2AX2/2 mice [40]. BLAST searches show good homology between Drosophila MU2 and human MDC1, and domain searches indicate that MU2 contains an N-terminal FHA domain, a C-terminal BRCT domain, and a region of concentrated TQ dipeptides, as found in MDC1. We have shown that the C-terminal BRCT domain of MU2 interacts with cH2Av, while the N-terminal FHA domain binds the MRN complex. MU2 co-localizes with cH2Av at repair foci in S2 cells and at recombination foci during meiosis. mu2a mutants are viable, and exhibit defects in meiotic recombination [18] and in the intra-S checkpoint, although they are not hypersensitive to killing by ionizing radiation or radiomimetic chemicals [17]. The lack of radiation sensitivity seen in mu2 mutants may be because we have not found a allele. Thus, by these measures MU2 appears to have the same structure and perform the same functions as MDC1. MU2 and the Generation of New Telomeres The notion that MU2 is an ortholog of MDC1 suggests that MU2 is necessary for an early step in lesion recognition and IRIF formation. Thus, DNA breaks induced in mu2 mutant oocytes would not be recognized properly. As a result, they would not induce cell cycle delay and would not be amenable to repair. Unrepaired broken chromosomes introduced into the zygote after completion of meiosis would then be subject to healing, i. e. the acquisition of a new telomere. In contrast, when males are irradiated and crossed to mu2a females, no terminal deficiencies were found, while fertility remained high. Thus, lesions in sperm seem to be repaired when placed in a mu2a mutant cytoplasm, and the effects of MU2 are limited to the oocyte nucleus. These PLoS Genetics | www.plosgenetics.org 8 May 2009 | Volume 5 | Issue 5 | e1000473