Unravelling the role of topoisomerase II beta binding protein 1 (TOPBP1) in the resolution of ultra-fine anaphase bridges.

在每次细胞分裂过程中，遗传密码必须在两个子细胞之间准确复制和分离。基因组的忠实分裂对于防止细胞分裂期间的损伤至关重要。控制这个过程的过程在整个进化过程中是保守的。此外，在复杂的多细胞生物体中，如人类，这些过程的准确性更加迫切，他们依赖于功能器官和组织之间的合作。在发育的早期阶段，这些机制的错误可能导致功能障碍或证明对生物体是致命的。尽管如此，我们目前对拓扑异构酶这类蛋白质解开和分离重复基因组的基本过程的理解仍然很差。不能解析缠结的DNA的细胞形成“染色质桥”，这些桥可能导致基因组信息的丢失或重排，从而导致染色体数量或结构的变化（染色体不稳定性）。这些桥可以根据它们被常规DNA染料染色的能力分为两个不同的类别，即非染色的“后期桥”或非染色的“超细后期桥”（UFB）。随后，染色体分离过程中的功能障碍通过癌症的发展或作为表现出终身发病率的遗传疾病的特征对人类健康构成挑战。因此，调节这些机制的基因是有效的肿瘤抑制剂，而靶向它们的药物是最成功的抗癌方式之一。总之，有迹象表明，染色体分离的进化保守机制是本质上的基础，对每个细胞的正常功能至关重要。为了深入了解这些机制，该项目的目的是探索蛋白质DNA拓扑异构酶II结合蛋白1（TOPBP 1）在招募DNA拓扑异构酶II α（TOP 2A）到UFB中的作用，从而导致它们在细胞分裂期间的解决。为了实现这一目标，将在科学家合作网络的帮助下解决三个研究目标。（一）。为了确定TOPBP 1招募到UFB的机制;与功能蛋白质组学小组的合作将有助于识别TOPBP 1相互作用蛋白质以及在UFB定位信号的修饰。将与ICR的基因功能团队合作，下调已鉴定的蛋白质相互作用物，以确定其对TOP 2A/TOPBP 1募集至UFB和细胞活力的影响。II）。为了阐明TOPBP 1-TOP 2A相互作用的分子基础;与ICR结构生物学部门合作，将有助于从细菌和/或昆虫细胞中纯化TOPBP 1和TOP 2A蛋白。这将使识别TOP 2A和TOPBP 1的相互作用表面和研究它们可能形成的复合物的结构成为可能。III）。确定这种相互作用如何促进UFB的TOP 2A招聘并帮助解决问题; ICR的光学显微镜设施将用于分析表达关键TOP 2A和TOPBP 1突变蛋白的正常和功能障碍细胞，以确定他们被招募到UFB的能力，并解决这些问题。拟议项目的结果将进一步加深我们对基本TOP 2A的理解-TOPBP 1过程在细胞分裂过程中调节基因组的忠实分裂，从而深入了解这些过程中的功能障碍如何影响人类健康，在该项目期间，将建立一个协作科学网络，以帮助获得广泛的实验技术方面的经验。这将是必不可少的，我的专业发展，成为一个主要的研究者谁将调查的分子机制，支持忠实的染色体分离在有丝分裂。

During every cell division, the genetic code must be accurately copied and separated between two daughter cells. Faithful division of the genome is vital to prevent damage during cell division. The processes which govern this process are conserved throughout evolution. Furthermore, there is a greater imperative for the accuracy of these processes in complex multicellular organisms, such as humans, who rely on the cooperation between functional organs and tissues. Errors in these mechanisms in the early stages of development may lead to dysfunction or prove lethal to the organism. Despite this, our current understanding of the fundamental processes that the class of proteins known as topoisomerases engage in to untangle and separate the duplicated genome remains poor. Cells that cannot resolve entangled DNA develop 'chromatin bridges' and these bridges may lead to the loss or rearrangement of genomic information leading to changes in the number or structure of chromosomes (chromosomal instability). These bridges can be divided into two distinct classes based on their ability to be stained with conventional DNA dyes i.e. stainable "anaphase bridges" or non-stainable "ultrafine anaphase bridges" (UFBs). Subsequently dysfunction in the processes of chromosomal separation pose a challenge to human health through development of cancer or as a feature of genetic diseases which demonstrate lifelong morbidity. In accordance, genes that regulate these mechanisms are potent tumour suppressors, whereas drugs that target them, are amongst the most successful anti-cancer modalities. Taken together, there is an indication that the evolutionarily conserved mechanisms of chromosomal segregation are fundamental in nature and essential to the normal function of every cell. To provide insight into these mechanisms the aim of this project is to explore the role of the protein DNA topoisomerase II binding protein 1 (TOPBP1) in recruiting the DNA topoisomerase II alpha (TOP2A) to UFBs leading to their resolution during cell division. To achieve this, three research objectives will be addressed with the aid of a collaborative network of scientists. I). To identify the mechanisms of recruitment of TOPBP1 to UFBs; Collaboration with the Functional Proteomics group, will aid in the identification of TOPBP1 interacting proteins and the modifications which signal their localisation at UFBs. Downregulation of identified protein interactors will be employed collaboration with the Gene Function Team at the ICR, to determine their impact on TOP2A/TOPBP1 recruitment to UFBs and viability of cells. II). To characterise the molecular basis of the TOPBP1-TOP2A interaction; collaboration with the Division of Structural Biology at the ICR, will assist in the purification of TOPBP1 and TOP2A proteins from bacteria and/or insect cells. This will enable the identification of the interacting surfaces of TOP2A and TOPBP1 and study of the structure of the complex that they may form. III). To determine how this interaction promotes TOP2A recruitment to UFBs and aids their resolution; The light microscopy facilities at the ICR will be used to analyse normal and dysfunctional cells that express key TOP2A and TOPBP1 mutant proteins, to determine their ability to be recruited to UFBs and resolve them.The findings of the proposed project will further our understanding of the fundamental TOP2A-TOPBP1 processes that regulate faithful division of the genome during cell division, providing insight into how dysfunction in these processes impacts on human health, leading to pathologies such as cancer.During this project a collaborative scientific network will be established which will aid in the acquisition of experience in a broad spectrum of experimental techniques. This will be essential for my professional development towards becoming a principal investigator who will investigate the molecular mechanisms that underpin faithful chromosomal segregation during mitosis.