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

点击翻译按钮获取中文摘要

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.