Regulation of chromosome segregation by SMC complexes and Top2 in S. pombe

描述(申请人提供)：由染色体结构维持(SMC)家族成员组成的三个多蛋白质复合体是染色体动力学的关键调节因子：粘附素(Smc1/3)、凝聚素(Smc2/4)和Smc5/6复合体。粘附素是姐妹染色单体凝聚力的关键决定因素，它被认为是形成环绕姐妹染色单体的环状结构。凝集素是有丝分裂染色体凝聚的关键，至少部分是通过产生超螺旋DNA来实现的。然而，也有数据将凝集素与染色体凝聚联系起来，并将凝聚蛋白与姐妹染色单体凝聚力联系起来。此外，这两个复合体都是DNA修复所必需的，这被认为是它们在染色体组织中所起作用的次要作用。此外，拓扑异构酶II(TOP2)除了在染色体脱落和有丝分裂染色体的支架形成中发挥明确的作用外，还具有凝聚和凝聚的功能。因此，当细胞在细胞周期中进行时，这些复合体之间存在着大量的串扰。第三个SMC复合体Smc5/6通过同源重组参与了DNA修复的晚期。然而，DNA修复中的作用是Smc5/6功能调节染色体结构的直接结果还是间接结果？它被描述为DNA修复复合体是有历史意义的，因为它最初是由裂殖酵母的辐射敏感突变体rad18-X(现在的smc6-X)定义的。此外，同一组rad突变体包括粘附素基因rad21。此外，Smc5/6对细胞活力是必不可少的，但DNA修复基因不是。因此，我们希望解决的中心问题是，为什么SMC5/6是必要的？到目前为止的数据表明，Smc5/6功能对于准确的染色体分离是必不可少的。我们之前已经证明，Smc5/6突变体显示出高水平的非整倍体，并在DNA损伤或TOP2功能障碍后显示失败的有丝分裂。此外，Smc5/6缺失突变体具有失败的有丝分裂的末端表型，没有外源性DNA损伤。我们认为所有这三个SMC复合体和TOP2在功能上相互作用，通过DNA复制和有丝分裂来指导染色体。虽然大多数关于Smc5/6的研究都是在DNA修复的背景下进行的，但本建议特别关注Smc5/6在染色体分离中的重要作用，以及Smc5/6如何与粘附素和拓扑异构酶II在功能上相互作用以确保染色体分离。为此，在S.pombe的工作中，我们追求采取细胞生物学、遗传学和生化方法的三个具体目标。首先，我们建立在大量初步数据的基础上，这些数据将DNA损伤后smc6突变体的有丝分裂缺陷与后期前期粘附素去除缺陷联系起来。在这里，我们讨论了因果关系和凝集素动力学的时空调节。其次，我们讨论了Smc5/6和TOP2之间的功能相互作用，我们认为这对未受损染色体的分离是至关重要的。最后，我们讨论了乙酰转移酶Eso1的功能，它是一种粘附素调节因子，将粘附素和Smc5/6功能机械地联系在一起。

DESCRIPTION (provided by applicant): Three multi-protein complexes scaffolded by members of the Structural Maintenance of Chromosomes (SMC) family of protein are key regulators of chromosome dynamics: cohesin (Smc1/3), condensin (Smc2/4) and the Smc5/6 complex. Cohesin is a key determinant of sister chromatid cohesion, where it is proposed to form a ring-shaped structure that encircles sister chromatids. Condensin is critical for mitotic chromosome condensation, at least in part by generating supercoiled DNA. However, there are also data to link cohesin to chromosome condensation, and condensin to sister chromatid cohesion. Further, both complexes are required for DNA repair, which is thought to be a secondary effect of their role in chromosome organization. Moreover, Topoisomerase II (Top2) functions in cohesion and condensation, in addition to its well-defined roles in chromosome decatenation and scaffolding of mitotic chromosomes. Thus, there is much cross talk between these complexes as cells progress through the cell cycle. The third SMC complex, Smc5/6, has been implicated in a late stage of DNA repair by homologous recombination. However, is a role in DNA repair a direct effect, or an indirect consequence of Smc5/6 function in regulating chromosome structure? Its description as a DNA repair complex is primarily historic, as it was first defined by rad18-X (now smc6-X), a radiation sensitive mutant in Schizosaccharomyces pombe. Moreover, the same collection of rad mutants includes the cohesin gene rad21. Further, Smc5/6 is essential for cell viability, but DNA repair genes are not. Thus the central question we wish to address is why is Smc5/6 essential? The data thus far suggest that Smc5/6 function is essential for accurate chromosome segregation. We have previously shown that Smc5/6 mutants show high levels of aneuploidy, and show failed mitoses following DNA damage or Top2 dysfunction. Further, Smc5/6 null mutants have a terminal phenotype of failed mitoses without extrinsic DNA damage. We propose that all three SMC complexes and Top2 functionally interact to guide chromosomes through DNA replication and mitosis. While most studies on Smc5/6 have been in the context of DNA repair, this proposal focuses specifically on the essential role of Smc5/6 in chromosome segregation, and how Smc5/6 functionally interacts with Cohesin and Topoisomerase II to ensure chromosome segregation. To this end, working in S. pombe, we pursue three specific aims that take cell biological, genetic, and biochemical approaches. First, we build on a significant body of preliminary data that links the mitotic defects of smc6 mutants following DNA damage to a defect in pre-anaphase cohesin removal. Here, we address cause versus consequence and the spatiotemporal regulation of cohesin dynamics. Secondly, we address the functional interaction between Smc5/6 and Top2, which we believe is critical for the segregation of undamaged chromosomes. Finally, we address the function of the acetyltransferase Eso1, a cohesin regulator that mechanistically links cohesin and Smc5/6 function.