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，对于生命和抑制人类疾病是必不可少的。基因组DNA被包装成蛋白质染色质，在染色质上发生转录、DNA复制和修复的关键过程。这些过程是由进化上保守的SMC家族推动的，其中包括粘附素(Smc1-Smc3)，它是姐妹染色单体凝聚和着丝粒功能所必需的；凝聚素(Smc2-Smc4)，需要拓扑异构酶II来紧凑并驱动有丝分裂时染色体的脱落；以及Smc5-Smc6复合体，其基本功能尚未确定(S)。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界面。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，这应该可以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复合体和由CDS1(HCHK2)强制实施的复制监视检查点之间的功能接口。我们的研究利用了遗传上易驯化的和经过充分验证的分裂酵母模式生物。对于我们下面的特定目标，分裂酵母是理想的，因为它有复杂的异染色着丝粒和相关的因子，这些在人类中是很保守的。此外，复制检查点的组件也高度保守。我们将通过将强大的酵母遗传学与尖端结构实验、生物化学和质谱学相结合来表征Smc5-Smc6全息复合体。我们有两个特定的目标，在这两个目标中，我们将确定SMC5-SMC6的有丝分裂和减数分裂染色体分离作用(S)及其相关的DNA修复蛋白Rad60。由于Rad60与Smc5-Smc6和复制检查点激酶CDS1在物理上相互作用，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界面在抑制有毒复制相关重组方面的生理重要性。此外，Rad60家族蛋白还含有串联相扑结构域(SLD)的独特结构特征。因此，我们将通过解决其SLD的结构来对Rad60进行功能表征，这将有助于突变结合体内表型分析。这些突变研究将确定RAD60在异染色质形成和稳定性中的作用机制(S)。在我们的第二个目标中，我们将表征Smc5-6复合体的有丝分裂和减数分裂同源重组修复和着丝粒功能。通过确定相扑E3连接酶Smc5-Smc6亚基Nse2的靶标，这将大大简化这一过程。这些研究将详细描述进化上保守的Smc5-Smc6、Rad60和复制检查点如何维持染色体的完整性，从而在人类中抑制疾病引发的基因组损伤。与公众健康相关我们已经确定了一种分子“机器”，它可以保护和修复细胞的“蓝图”或基因组。基因组包含维持细胞正常生长所需的信息，如果这些信息被破坏，癌症可能会接踵而至。我们的研究将为细胞生长的正常调控提供关键的见解，并进一步揭示癌细胞的潜在“阿喀琉斯跟腱”。

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.