Chromatin Structure and DNA Repair

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Previously, we discovered mutations in a gene mu2 in Drosophila that allow the recovery of chromosome aberrations that have lost a natural telomere and regained a structure that protects the chromosome end. These neotelomeres have lost the DNA motifs normally associated with telomeres, but retain the proteins that protect the chromosome ends and distinguish them from chromosome breaks. Recently, we found that the MU2 protein is distributed along chromosome arms in the absence of DNA damage, but upon radiation induced damage to DNA redistributes to the radiation-induced repair foci. The protein is also found at sites of meiotic recombination, which is induced by DNA double strand breaks. When present in these foci, MU2 acts as a scaffold, with one end of the protein binding to a complex of the repair proteins MRE11, RAD50 and NBS and the other end to a phosphorylated form of variant histone H2Av known as gammaH2Av. (Drosophila H2Av is an ortholog of human H2AX.) Based on sequence alignments, domain structure and protein function, MU2 appears to be an ortholog of the human MDC1 protein. Mutations in the mu2 gene cause a decrease in the number and size of radiation induced repair foci and meiotic recombination foci. The rate of DNA repair appears to be reduced, although repair is not blocked. Similarly, cell cycle regulation in response to DNA damage is decreased in these mutants, but not blocked entirely. In an attempt to understand the interaction of factors that control telomere stability and chromatin structure, we are looking for chromatin proteins that interact with MU2. One of these is heterochromatin protein 1 (HP1a), which binds to MU2 in the absence of DNA damage. After radiation treatment, MU2 is drawn to repair foci, while at the same time the foci are removed from nuclear regions that are rich in HP1a, suggesting that the association between MU2 and HP1 is broken when repair foci form. We are also making double mutation combinations with a mutation in the mu2 gene along with a mutation that decreases telomere stability and increases telomere fusions. These latter mutations are in genes such as mre11, rad50, nbs and Su(var)205, the gene that encodes HP1a. We will test both the generation of new telomeres and the frequency of telomere fusions in these double mutant lines.