Chromatin Structure and DNA Repair

此前，我们在果蝇中发现了基因mu2的突变，该突变允许染色体畸变的恢复，这些染色体畸变已经失去了天然端粒并重新获得了保护染色体末端的结构。这些新端粒失去了通常与端粒相关的DNA基序，但保留了保护染色体末端并将其与染色体断裂区分开来的蛋白质。最近，我们发现MU2蛋白在没有DNA损伤的情况下沿着染色体臂分布，但在辐射诱导的DNA损伤后重新分布到辐射诱导的修复灶。该蛋白也存在于减数分裂重组的位点，这是由DNA双链断裂诱导的。当存在于这些病灶中时，MU2充当支架，其中蛋白质的一端与修复蛋白MRE 11、RAD 50和NBS的复合物结合，另一端与称为γ H2Av的变体组蛋白H2Av的磷酸化形式结合。（果蝇H2Av是人类H2AX的直系同源物。基于序列比对、结构域结构和蛋白质功能，MU2似乎是人MDC 1蛋白的直系同源物。mu2基因突变导致辐射诱导的修复灶和减数分裂重组灶的数量和大小减少。DNA修复的速率似乎降低，尽管修复没有被阻断。类似地，在这些突变体中，响应于DNA损伤的细胞周期调节降低，但不完全阻断。 为了了解控制端粒稳定性和染色质结构的因素之间的相互作用，我们正在寻找与MU2相互作用的染色质蛋白。其中之一是异染色质蛋白1（HP1a），它在没有DNA损伤的情况下与MU2结合。放射治疗后，MU 2被吸引来修复病灶，同时病灶从富含HP 1a的核区域被去除，这表明当修复病灶形成时，MU 2和HP 1之间的联系被打破。我们也在制造双重突变组合，其中mu2基因的突变沿着降低端粒稳定性和增加端粒融合的突变。这些突变发生在mre 11、rad50、nbs和Su（var）205等基因中，后者是编码HP1a的基因。我们将在这些双突变株系中测试新端粒的产生和端粒融合的频率。

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.