Establishment of sister chromatid cohesion

描述（由申请人提供）：在染色体分离、DNA修复和转录过程中，染色体被组织成形态上不同的高级结构。这些不同的结构是由染色体结构维持（smc）复合物介导的，Smc复合物由从细菌到人保守的4个蛋白质亚基组成。Smc复合物被认为将染色质的两个不同区域拴在一起，无论是在同一DNA链内还是在两个不同链之间。然而，它们如何结合染色质或将其系留的机制仍然知之甚少。一种Smc复合物，粘着蛋白，将两个姐妹染色单体（新复制的染色体）从它们在S期合成的时间到有丝分裂中期保持在一起。这种姐妹染色单体的凝聚力对于确保子细胞在细胞分裂过程中继承适当的倍性至关重要。粘着蛋白对有丝分裂凝聚、减数分裂染色体结构、复制后DNA修复和基因表达调控也很重要。粘连蛋白和粘连蛋白辅助因子的缺陷是几种人类疾病的原因，并可能导致与肿瘤发生相关的染色体不稳定性。在本提案中，我概述了一种综合方法，利用遗传学、细胞生物学和新的体外生化测定来研究使用芽殖酵母建立凝聚力。我们将使用一种新的系统来产生总细胞粘附素连续量化减少的菌株。这些菌株的分析将提供深入了解的水平和本地化的凝聚素，需要在体内介导其已知的不同的作用，在染色体代谢，以及发现潜在的新的生物功能。我们将研究的作用，ATP，粘附素加载和粘附素拮抗剂在粘附素结合到染色体，利用显性负表型的粘附素的Smc 3 ATP酶和抑制分析其有缺陷的加载。我们将阐明染色质结合的粘着蛋白转换为其凝聚状态的机制。体内实验将证明粘附素亚基对不同于染色质结合的粘附产生的贡献。我们还将测试内聚性是否反映了姐妹染色单体周围的内聚环的稳定性或与每个姐妹染色单体结合的内聚复合物之间的寡聚化。我们还将通过我们最近开发的与染色质结合的体外生理相关测定来分析粘着蛋白与染色体结合的机制。使用这种方法，我们将评估DNA序列，DNA拓扑结构，ATP，cohesin本身，和cohesin辅助因子的cohesin-DNA复合物的形成的贡献。我们将开发一个相关的体外测定凝聚力作为基础，我们在体外组装的凝聚素-DNA复合物和我们的电池的凝聚素突变。最后，组装在体外的cohesin/DNA复合物和粘性cohesin/DNA复合物的能力将使我们能够确定它们的超微结构在电子显微镜下。 公共卫生相关性：DNA，染色体的遗传物质，首先被核心组蛋白包裹，然后被称为SMC复合物的特殊蛋白质复合物包装成更高级的结构，就像磁带中的磁带被塑料外壳包装一样。Smc复合物介导的染色体结构确保生物体中每个新形成的细胞继承完整的染色体，并且染色体DNA的损伤可以有效修复。编码Smc蛋白的人类基因突变与癌症、出生缺陷和疾病有关。

DESCRIPTION (provided by applicant): Chromosomes are organized into morphologically distinct higher-order structures during chromosome segregation, DNA repair, and transcription. These different structures are mediated by structural maintenance of chromosome (smc) complexes that consist of 4 protein subunits conserved from bacteria to man. Smc complexes are thought to tether together two distinct regions of chromatin, either within the same DNA strand or between two distinct strands. However the mechanism for how they bind chromatin or tether it, are still very poorly understood. One Smc complex, cohesin, holds together the two sister chromatids (the newly replicated chromosomes) from the time of their synthesis in S phase through metaphase of mitosis. This sister chromatid cohesion is critical to ensure that daughter cells inherit proper ploidy during cell division. Cohesin is also important for mitotic condensation, meiotic chromosome structure, post-replicative DNA repair and the regulation of gene expression. Defects in cohesin and cohesin auxiliary factors are the cause of several human disorders and potentially contribute to chromosome instability associated with tumorigenesis. In this proposal I outline an integrated approach using genetics, cell biology, and new in vitro biochemical assays to study the establishment of cohesion using budding yeast. We will use a novel system to generate strains with serial quantized reduction in total cellular cohesin. Analysis of these strains will provide insights into the level and localization of cohesin that is needed in vivo to mediate its known diverse roles in chromosome metabolism as well as uncover potentially novel biological functions. We will investigate the role of ATP, cohesin loader and a cohesin antagonist in cohesin binding to chromosomes by exploiting dominant negative phenotypes of cohesin's Smc3 ATPase and by suppression analysis of its defective loader. We will elucidate the mechanism of converting chromatin bound cohesin to its cohesive state. Experiments in vivo will demonstrate the contribution of cohesin subunits to cohesion generation distinct from chromatin binding. We will also test whether cohesiveness reflects stabilization of the cohesin ring around the sister chromatids or oligomerization between cohesin complexes bound to each sister chromatid. The mechanism of cohesin binding to chromosomes will also be analyzed by a physiological-relevant in vitro assay for cohesin binding to chromatin that we have developed recently. Using this assay we will assess the contribution of DNA sequence, DNA topology, ATP, cohesin itself, and cohesin auxiliary factors to the cohesin-DNA complex formation. We will develop a relevant in vitro assay for cohesion using as a foundation our in vitro assembled cohesin-DNA complexes and our battery of cohesin mutations. Finally, the ability to assemble in vitro cohesin/DNA complexes and cohesive cohesin/DNA complexes will allow us to determine their ultrastructure in the electron microscope. PUBLIC HEALTH RELEVANCE: DNA, the hereditary material of chromosomes, is coated first by core histone proteins and then packaged into higher order structures by specialized protein complexes, called SMC complexes, much the way the magnet tape in a cassette is packaged by its plastic housing. Chromosome structures mediated by Smc complexes ensure that each newly form cell in an organism inherits the complete complement of chromosomes and that damage to the DNA of the chromosomes can be repaired efficiently. Mutations in human genes encoding Smc proteins have been linked to cancer, birth defects and disorders.