Roles of the Smc5-Smc6 Holocomplex in Genome Stability

描述（由申请人提供）：在细胞分裂时将未受损的染色体忠实地传递给子细胞是生命和抑制人类疾病所必需的。基因组DNA被包装成蛋白质染色质，转录、DNA复制和修复的关键过程都发生在染色质上。这些过程是由进化上保守的SMC家族促进的，其中包括姐妹染色单体内聚和着丝粒功能所需的内聚蛋白（Smc1-Smc3）；凝聚蛋白（Smc2-Smc4），与拓扑异构酶II一起在有丝分裂时紧密化和驱动染色体的十烷化；以及Smc5-Smc6复合体，其基本功能尚不明确。我们的长期目标是剖析和定义DNA修复、复制和细胞周期进展机制之间的关键调控接口。最终，这将产生对这些过程之间相互作用的分子水平的理解，这将解释许多人类疾病的病因，并可能突出潜在的治疗策略。目前，我们正在定义Smc5-Smc6复合体和复制监视检查点之间的功能接口，由Cds1 （hCHK2）执行。我们的研究利用了遗传上易于处理和得到充分证明的裂变酵母模型生物。对于我们下面的具体目标，裂变酵母是理想的，因为它具有复杂的异色着丝粒和相关因子，在人类中保存得很好。此外，复制检查点的组件也是高度保守的。我们将通过整合强大的酵母遗传学与尖端的结构实验，生物化学和质谱来表征Smc5-Smc6全息复合物。我们有两个特定的目的，我们将确定Smc5-Smc6和相关的DNA修复蛋白Rad60的有丝分裂和减数分裂染色体分离作用。由于Rad60与Smc5-Smc6和复制检查点激酶Cds1相互作用，它在复制检查点和DNA修复过程之间提供了一个接口。在我们的第一个目标中，我们将测试Cds1-Rad60界面在抑制毒性复制相关重组中的生理重要性。此外，Rad60家族蛋白含有独特的串联sumo样结构域（SLDs）结构特征。因此，我们将通过求解其SLDs的结构来对Rad60进行功能表征，这将有助于突变和体内表型分析。这些诱变研究将确定Rad60介导其在异染色质形成和稳定性中所观察到的作用的机制。在我们的第二个目标中，我们将描述Smc5-6复合物的有丝分裂和减数分裂同源重组修复和着丝粒功能。通过确定SUMO E3连接酶Smc5-Smc6亚基Nse2的靶标，这将大大促进这一点。这些研究将对进化上保守的Smc5-Smc6、Rad60和复制检查点维持染色体完整性的方式产生详细的了解，从而在人类中抑制疾病引发的基因组损伤。我们已经确定了一种保护和修复细胞“蓝图”或基因组的分子“机器”。基因组包含维持正常细胞生长所需的信息，如果这些信息被破坏，癌症就会随之而来。我们的研究将为细胞生长的正常调节提供关键的见解，进一步揭示癌细胞的潜在“阿喀琉斯之踵”。

DESCRIPTION (provided by applicant): The faithful transmission of undamaged chromosomes to daughter cells at cell division is essential for life and for suppression of human disease. Genomic DNA is packaged into proteinaceous chromatin, upon which the crucial processes of transcription, DNA replication and repair occur. These processes are facilitated by the evolutionarily conserved SMC family, which includes cohesin (Smc1-Smc3), required for sister chromatid cohesion and centromere function; condensin (Smc2-Smc4), required with topoisomerase II to compact and drive the decatenation of chromosomes at mitosis; and the Smc5-Smc6 complex, whose essential function(s) are undefined. Our long-term objective is to dissect and define critical regulatory interfaces between the DNA repair, replication and cell cycle progression mechanisms. Ultimately, this will yield a molecular level understanding of the interplay between these processes that should explain the etiology of many human diseases and perhaps highlight potential therapeutic strategies. Currently, we are defining the functional interface between the Smc5-Smc6 complex and the replication monitoring checkpoint, enforced by Cds1 (hCHK2). Our studies utilize the genetically tractable and well-proven fission yeast model organism. For our Specific Aims below, fission yeast is ideal, as it has complex heterochromatic centromeres and associated factors that are well conserved in humans. Furthermore, components of the replication checkpoint are also highly conserved. We will characterize the Smc5-Smc6 holocomplex by integrating powerful yeast genetics with cutting-edge structural experiments, biochemistry and mass spectrometry. We have two Specific Aims in which we will determine the mitotic and meiotic chromosome segregation role(s) of Smc5-Smc6 and the associated DNA repair protein, Rad60. As Rad60 physically interacts with both Smc5-Smc6 and the replication checkpoint kinase Cds1, it provides an interface between the replication checkpoint and DNA repair processes. In our first Aim we will test the physiological importance of the Cds1-Rad60 interface in suppressing toxic replication-associated recombination. In addition, Rad60 family proteins contain the unique structural signature of tandem SUMO-like domains (SLDs). Therefore, we will functionally characterize Rad60 by solving the structures of its SLDs, which will facilitate mutagenesis coupled with in vivo phenotypic analyses. These mutagenesis studies will define the mechanism(s) by which Rad60 mediates its observed role in heterochromatin formation and stability. In our second Aim we will characterize the mitotic and meiotic homologous recombination repair and centromeric functions of the Smc5-6 complex. This will be greatly facilitated by identifying targets of the SUMO E3 ligase Smc5-Smc6 subunit Nse2. These studies will yield a detailed picture of the ways in which the evolutionarily conserved Smc5-Smc6, Rad60 and replication checkpoint maintain chromosome integrity and thus in humans, suppress disease priming genomic lesions. PUBLIC HEALTH RELEVANCE We have identified a molecular "machine" that protects and repairs the cells "blueprint" or genome. The genome contains information required to maintain normal cell growth and if this information is damaged, cancer can ensue. Our studies will provide key insights on normal regulation of cell growth and furthermore, reveal a potential "Achilles' Heel" of cancer cells.