Chromatin Structure and DNA Repair

此前，我们发现了果蝇mu 2基因的突变，这使得染色体畸变的恢复失去了天然端粒，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MU 2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。该蛋白质也积累在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，MU 2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 H2 Av的变体组蛋白H2 Av的磷酸化形式结合。（果蝇H2 Av是人类H2 AX的直系同源物。基于序列比对、结构域结构和蛋白质功能，MU 2似乎是人MDC 1蛋白的直系同源物。mu 2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MU 2相互作用的染色质蛋白。其中之一是异染色质蛋白1（HP 1a），它在没有DNA损伤的情况下与MU 2结合。放射治疗后，MU 2被吸引到整个核中修复病灶。修复灶在异染色质中形成，异染色质是富含HP 1a的浓缩染色质，在处理的几秒钟内，然后它们从异染色质移到具有更松弛的常染色质的核结构域。RNAi敲低HP 1a阻止了异染色质中病灶的去除，并增加了G2/M期阻滞和细胞凋亡。这些观察结果表明，DNA修复需要一个放松的染色质结构进行。 在果蝇中，将复合体结合在一起的支架蛋白也与调节卵子发生时间的蛋白质相互作用。当我们使用突变减少MU 2蛋白的量时，一种调节蛋白ORB（哺乳动物细胞质聚腺苷酸化元件结合蛋白（CPEB）的果蝇同源物）的水平也降低，下游效应蛋白也是如此。因此，染色体事件的进展相对于卵子发生的细胞事件暂时变得延迟，尽管突变的卵母细胞最终恢复并且突变的雌性是可育的。这些结果与减数分裂期间染色体事件与卵子发生的发育进程之间存在通信以保持它们同步的想法一致。

Previously, we discovered mutations in the mu2 gene in the fruit fly, 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 also accumulates at sites of meiotic recombination, which are 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 throughout the nucleus. Repair foci form in heterochromatin, which is HP1a rich, condensed chromatin, within a few seconds of treatment, then they are removed from heterochromatic to nuclear domains with more relaxed euchromatin. RNAi knockdown of HP1a prevents the removal of foci from heterochromatin, and increases G2/M arrest and apoptosis. These observations suggest that DNA repair requires a relaxed chromatin structure to proceed. The scaffold protein that holds the complex together in Drosophila also interacts with proteins that regulate the timing of oogenesis. When we reduce the amount of MU2 protein using a mutation, the level of one regulatory protein ORB, a Drosophila homolog of mammalian cytoplasmic polyadenylation element binding protein (CPEB), is also decreased, as are down stream effector proteins. The progression of chromosomal events thus temporarily becomes delayed relative to the cellular events of oogenesis, although the mutant oocytes eventually recover and mutant females are fertile. These results are consistent with the idea that there is communication between chromosomal events during meiosis and developmental progression of oogenesis in order to keep them in synch.