How does Condensin mediate topological change during mitosis?

保持基因组的稳定对于正常的细胞功能至关重要。导致遗传信息丢失或复制的细胞缺陷可能导致细胞死亡、癌症或过早衰老。在细胞分裂过程中，维持基因组稳定性是一个特别尖锐的问题。每次细胞分裂时，组成基因组的每一条染色体都必须被完美复制。然后，两个拷贝必须从彼此中完全分解，最后每个染色体的一个拷贝被输送到每个子细胞。如果不能忠实地完成任何一个或这些步骤，可能会对基因组的稳定性造成灾难性的影响。因此，了解细胞蛋白质如何协同工作来维持基因组的不稳定性，对于理解细胞为什么会随着年龄的增长而变得癌症或缺陷至关重要。SMC蛋白家族似乎对从细菌到人类的所有生物的基因组稳定性至关重要。SMC蛋白在细胞中形成几种不同的复合体，但在所有情况下，它们似乎都通过影响染色体结构来发挥作用。其中一种称为凝集素的SMC复合体似乎对有丝分裂中的染色体紧凑至关重要。凝聚素如何压缩染色体仍然是个谜，尽管它已经在试管中显示出在DNA中产生超螺旋的“弹簧状”结构。最近我发现，在染色体分离之前，细胞中也形成了类似的结构。染色体结构的这种变化促进了染色体的分辨率，因此对染色体的稳定至关重要。本提案的工作旨在说明凝聚素复合体是如何改变染色体结构以促进基因组稳定的。它将首先使用分子生物学技术来修改凝集素因子SMC2和SMC4的不同区域。然后，我们将使用这些修饰的蛋白质来评估SMC2和SMC4的特定功能是如何在细胞中产生超螺旋结构所必需的。然后，改变后的蛋白质产生超螺旋结构的能力将与凝聚素如何改变染色体组织以及细胞确保每个染色体的一个完整副本被分离到每个子细胞的能力有关。这些实验的目的是提供详细的描述，说明细胞内的染色体结构需要哪些方面的SMC/凝聚素功能来改变。然后，我们将使用这些信息在试管中重新创造这种情况，从而产生一个系统，在这个系统中，我们将能够充分描述凝聚素是如何实现其功能的。使用这种方法的组合，这项工作旨在为SMC/凝聚素蛋白质，以及潜在的所有其他SMC蛋白质如何帮助维持基因组稳定提供独特的见解。该项目产生的信息不仅对研究基因组稳定性的研究人员普遍感兴趣，而且对衰老和癌症进化的研究也有普遍意义。

Maintaining genome stability is essential for normal cellular function. Cellular defects that lead to loss or duplication of genetic information can lead to cell death, cancer or premature aging. Maintenance of genome stability is a particularly acute problem in dividing cells. Every time a cell divides, every one of the chromosomes that makes up the genome must be perfectly copied. The two copies must then fully resolved from one other and finally one copy of every chromosome transported into each daughter cell. Failure to faithfully complete any or these steps could have disastrous effects on genome stability.Understanding how cellular proteins work together to maintain genome instability is therefore crucial to understanding why cells become cancerous or defective with age. The SMC family of proteins appear crucial to genome stability in all organisms from bacteria to humans. The SMC proteins form several different complexes in the cell but in all cases they appear to function by influencing chromosome structure.One of the SMC complexes called condensin appears to be important for chromosomal compaction in mitosis. How condensin compacts chromosomes remains enigmatic although it has been shown in the test tube to generate supercoiled "spring like" structures in DNA. Recently I have shown that similar structures are formed in cells just before the chromosomes are segregated. This change in chromosome structure promotes the resolution of the chromosomes and so appears crucial for chromosome stability.The work in this proposal aims to demonstrate how the condensin complex changes chromosome structure in order to promote genome stability. It will first use molecular biology techniques to modify distinct regions of the condensin factors SMC2 and SMC4. We will then use these modified proteins to assess how specific aspects of SMC2 and SMC4 function are required to generate supercoiled structures in the cells. The ability of the altered proteins to generate supercoiled structures will then be related to how condensin alters chromosome organisation and the cells ability to ensure that one whole copy of each chromosome is segregated to each daughter cell. These experiments aim to provide a detailed description of what aspects of SMC/condensin function are required to change chromosome structure inside cells. We will then use this information to recreate the situation in a test tube and so generate a system where we will be able to fully describe how condensin achieves its function.Using this combination of approaches this work aims to provide unique insights into how SMC/condensin proteins, and potentially all other SMC proteins help maintain genome stability. The information generated by this project will be of general interest not only to researchers studying genome stability but also for research into ageing and cancer evolution.