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学上不同的高阶结构。这些不同的结构是由染色体（smc）复合体的结构维持介导的，该复合体由4个从细菌到人类保守的蛋白质亚基组成。Smc复合体被认为将染色质的两个不同区域连接在一起，要么在同一条DNA链内，要么在两条不同的DNA链之间。然而，它们如何结合或系住染色质的机制仍然知之甚少。一个Smc复合体，内聚蛋白，将两个姐妹染色单体（新复制的染色体）从S期合成到有丝分裂中期连接在一起。这种姐妹染色单体内聚对确保子细胞在细胞分裂过程中继承适当的倍性至关重要。内聚蛋白在有丝分裂凝聚、减数分裂染色体结构、复制后DNA修复和基因表达调控中也很重要。粘接蛋白和粘接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。在这个提议中，我概述了一个综合的方法，利用遗传学，细胞生物学和新的体外生化分析来研究利用芽殖酵母建立凝聚力。我们将使用一种新的系统来产生具有连续量化总细胞内聚蛋白减少的菌株。对这些菌株的分析将提供对体内所需的内聚蛋白水平和定位的见解，以介导其在染色体代谢中的已知多种作用，并揭示潜在的新生物学功能。我们将通过利用黏结蛋白Smc3 ATP酶的显性负表型和对其缺陷装载物的抑制分析，研究ATP、黏结蛋白装载物和黏结蛋白拮抗剂在黏结蛋白与染色体结合中的作用。我们将阐明将染色质结合的内聚蛋白转化为其内聚状态的机制。体内实验将证明内聚蛋白亚基对内聚产生的贡献不同于染色质结合。我们还将测试内聚性是否反映了姐妹染色单体周围内聚蛋白环的稳定性或与每个姐妹染色单体结合的内聚蛋白复合物之间的寡聚化。黏结蛋白与染色体结合的机制也将通过我们最近开发的黏结蛋白与染色质结合的生理相关体外试验来分析。利用这种分析方法，我们将评估DNA序列、DNA拓扑结构、ATP、内聚蛋白本身和内聚蛋白辅助因子对内聚-DNA复合物形成的贡献。我们将开发一种相关的体外内聚分析方法，使用我们体外组装的内聚- dna复合物和内聚蛋白突变电池作为基础。最后，在体外组装内聚蛋白/DNA复合物和内聚内聚蛋白/DNA复合物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。

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.