Regulation of chromosome cohesion during cell cycle progression

在后生动物中，粘附素复合体在基因组结构的许多基本方面和 维修。它确保了细胞过程中DNA复制产物的准确排列和分离 通过在它们形成时将它们捆绑在一起来进行分裂。它还促进染色体的正常包装进入 DNA复制前后的间期核，进而确保适当的基因调控。 最后，在零星和程序性DNA双链断裂之后，粘附素促进DNA修复。但 如何控制和整合粘附素复合体的这些不同的活动，以确保 顺序和结构的完整性？这一基本问题是本提案中实验的基础。 粘附素与染色质的联系是动态的：特定时间和地点的粘附素活动反映了 支持和反对凝聚力活动的集体结果。凝集素稳定性或染色质的变化 结合发生在对细胞周期进程、DNA损伤信号的响应，以及对某些 发育线索。然而，在许多情况下，导致粘附素结合变化的信号通路 人们对此了解甚少。Eco1家族成员对粘附素Smc3亚单位的修饰 乙酰转移酶稳定粘附素与染色质的相互作用。在脊椎动物中，有两个成员 在这个家族中，Eco1和ESCO2在凝集素调节中扮演着明显不同的角色。 ESCO2是在DNA复制过程中建立姐妹染色单体凝聚力所必需的，而ESCO1则是调节的 粘附素在促进正常染色体结构中的作用。通过这里描述的实验，我们将 定义这两种酶是如何被调节的，以使用相似的催化活性产生非常不同的结果。 在这个提案中，我描述了一系列使用细胞培养模型、生物化学和功能 分析非洲爪哇卵提取液以确定Eco1和ESCO2是如何被调节以执行其独特的 功能。在目标1中，我们将定义ESCO2与复制的关联的分子基础 仪器。在目标2和目标3中，我们将定义Eco1是如何调节染色质结构的，以及 这项关于DNA修复的规定。在目标4中，我们将通过分析Eco1对染色体结构的影响来定义 G1期的核组装。这些实验将从机制上详细阐明粘附素的功能 通过DNA复制和细胞周期进展来确保适当的姐妹染色单体连接和 间期核的功能基因组图谱。

In metazoans, the cohesin complex plays critical roles in many fundamental aspects of genome structure and maintenance. It ensures the accurate alignment and segregation of the products of DNA replication during cell division by tethering them together as they are made. It also promotes normal packaging of chromosomes into the interphase nucleus, both before and after DNA replication, which in turn ensures proper gene regulation. Finally, cohesin promotes DNA repair following both sporadic and programmed DNA double strand breaks. But how are these distinct activities of the cohesin complex controlled and integrated to ensure maintenance of sequence and structural integrity? This fundamental question is the basis for the experiments in this proposal. The association of cohesin with chromatin is dynamic: cohesin activity at a particular time and place reflects the collective outcome of both pro- and anti-cohesive activities. Changes in cohesin stability or chromatin binding occur in response to cell cycle progression, DNA damage signaling, and in response to certain developmental cues. In many cases, however, the signaling pathways that result in changes in cohesin binding are poorly understood. Modification of the Smc3 subunit of cohesin by members of the Eco1 family of acetyltransferases stabilizes the interaction of cohesin with chromatin. In vertebrates, there are two members of this family, Esco1 and Esco2, which we have shown play distinctly different roles in cohesin regulation. Esco2 is essential for establishing sister chromatid cohesion during DNA replication, while Esco1 modulates cohesin in its role in promoting normal chromosome architecture. With the experiments described here we will define how these two enzymes are regulated to generate very different outcomes using similar catalytic activity. In this proposal I describe a series of experiments using cell culture models, biochemistry, and functional analysis in Xenopus egg extracts to define how Esco1 and Esco2 are regulated to perform their unique functions. In Aim 1, we will define the molecular basis for the association of Esco2 with the replication apparatus. In Aims 2 and 3, we will define how chromatin structure is regulated by Esco1, and the impact of this regulation on DNA repair. In Aim 4 we will define how Esco1 impacts chromosome structure by analyzing nuclear assembly in G1. These experiments will elucidate in mechanistic detail how cohesin function is entrained by DNA replication and cell cycle progression to ensure proper sister chromatid tethering and a functional genomic landscape in interphase nuclei.