Establishment of sister chromatid cohesion

描述(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)复合体介导的，SMC复合体由4个蛋白质亚基组成，从细菌到人类都是保守的。SMC复合体被认为将染色质的两个不同区域捆绑在一起，要么在同一条DNA链内，要么在两条不同的链之间。然而，它们如何结合或束缚染色质的机制仍然知之甚少。粘附素是一种SMC复合体，从S期合成到有丝分裂中期，它将两个姐妹染色单体(新复制的染色体)连接在一起。这种姐妹染色单体凝聚力对于确保子代细胞在细胞分裂过程中继承适当的倍性是至关重要的。粘附素对有丝分裂凝聚、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。粘附素和粘附素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。在这项建议中，我概述了一种综合的方法，使用遗传学、细胞生物学和新的体外生化分析来研究利用发芽酵母建立凝聚力。我们将使用一种新的系统来产生细胞粘附素总量连续量化减少的菌株。对这些菌株的分析将提供对体内粘附素水平和定位的洞察，粘附素是介导其在染色体代谢中的已知不同角色以及揭示潜在的新的生物学功能所需的。我们将通过利用粘附素的Smc3 ATPase的显性负性表型和对其缺陷加载器的抑制分析来研究ATP、粘附素加载器和粘附素拮抗剂在粘附素与染色体结合中的作用。我们将阐明染色质结合的粘附素转化为其内聚状态的机制。体内实验将证明，与染色质结合不同，粘附素亚基对凝聚力产生的贡献不同。我们还将测试凝聚力是否反映了姐妹染色单体周围粘附素环的稳定，或者反映了与每个姐妹染色单体结合的粘附素复合体之间的寡聚。我们还将通过我们最近开发的与生理相关的体外粘附素与染色质结合的方法来分析粘附素与染色体结合的机制。利用这一方法，我们将评估DNA序列、DNA拓扑结构、ATP、粘附素本身以及粘附素辅助因素对粘附素-DNA复合体形成的贡献。我们将以我们的体外组装的粘附素-DNA复合体和我们的粘附素突变组为基础，开发一种相关的体外凝聚力测试方法。最后，在体外组装粘附素/DNA复合体和粘附性粘附素/DNA复合体的能力将使我们能够在电子显微镜下确定它们的超微结构。 与公共健康相关：DNA是染色体的遗传物质，它首先被核心组蛋白包裹，然后由被称为SMC复合体的特殊蛋白质复合体包装成更高阶的结构，就像盒式磁带的塑料外壳包装磁带一样。SMC复合体介导的染色体结构确保了生物体中每个新形成的细胞都继承了完整的染色体，并确保了染色体DNA的损伤能够得到有效的修复。人类编码SMC蛋白的基因突变与癌症、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.