Investigating the mitotic roles of Topoisomerase 2 Alpha and Topoisomerase 2 Beta-Binding Protein 1 in the maintenance of chromosome structure and genome stability

Type II Topoisomerases are essential, evolutionarily conserved proteins, important throughout the cell cycle. They act by generating a transient double-stranded DNA break in one molecule and manoeuvring a second DNA segment through the resultant gap. This ability to catenate and decatenate DNA means that type II topoisomerases are important not only in relieving DNA supercoiling during DNA transcription, replication, repair and recombination, but also in chromosome compaction and segregation during mitosis. In vertebrates, two isoforms of type IIA topoisomerase exist: topoisomerase 2 alpha (TOP2A) and topoisomerase 2 beta (TOP2B). Most differences reside in the C-terminal regions, which are crucial in determining isoform-specific functions. Topoisomerase 2 beta-binding protein 1 (TOPBP1) is conserved throughout eukaryotes and has nine BRCT (BRCA1 C-terminus) domains which allow it to interact dynamically with various phospho-proteins. It was originally identified, through a yeast two-hybrid screen, as a factor interacting with TOP2B. It has since been reported to interact with TOP2A during anaphase. However, most functions of TOPBP1 appear to be independent of Topoisomerase 2 (TOP2), with it acting as a multifunctional scaffold, recruiting factors to the genome during transcription, replication, and the DNA damage response. Recently, a role for TOPBP1 in M phase has also emerged. In the first part of this thesis, I focus on the role of TOP2 in orderly metaphase congression and the maintenance of chromosome compaction during prometaphase-metaphase. These studies have been undertaken in human tumour-derived cell lines using both chemical inhibition and depletion (through siRNA and genetically modified cell lines) to disrupt the two isoforms. My findings indicate that, in cells in which chromosome compaction has already occurred (prometaphase-metaphase), either chemical inhibition of the enzyme (ICRF-193 or merbarone), or acute protein degradation (AID system) of TOP2A, results in some loss of chromosome compaction. An in vitro assay of metaphase chromosome structure suggests that acute removal of TOP2A protein, from pre-condensed chromosomes, perturbs their ability to refold. Taken together, these results suggest TOP2A has an ongoing role in mitotic chromosome maintenance during prometaphase-metaphase, both through innate enzymatic activity and as a protein linker, contributing to structural integrity and memory. Studies into the disorderly nature of metaphase congression, following depletion of TOP2A from cycling cells, led me to investigate Topoisomerase 2 Beta Binding Protein 1 (TOPBP1). Thus, the second part of my thesis focuses on this protein’s interactions with Mediator of DNA Damage Checkpoint 1 (MDC1) and Cancerous Inhibitor of Protein Phosphatase 2 A (CIP2A) and their interdependencies. Using siRNA and the AID system to knock down levels of the three proteins individually, I have examined the impact of those disruptions on the recruitment of TOPBP1 to the chromatin and centrosomes during M phase. Finally, I have used transient co-transfection assays to test the effect of mutating specific residues in full-length TOPBP1 on its localisation at the mitotic centrosome. I show that a K154A/K155A mutated version of TOPBP1, reported by others to disrupt the protein’s interaction with MDC1 and recruitment to mitotic chromatin, increased the frequency of TOPBP1-positive metaphase centrosomes. This was consistent with my finding using siMDC1. Conversely, I report that K1317A interferes with the centrosomal recruitment of TOPBP1. In summary, I find evidence supporting an ongoing role for TOP2A in the maintenance of mitotic chromosome structure. In addition, my findings on the localisation of TOPBP1 at the metaphase centrosome raise the possibility that TOPBP1 contributes to genome stability not only through its recruitment to sites of DNA damage during M phase, but also through an under explored role at the mitotic centrosome.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Benoit, Judith

Advisor dc:contributor.advisor

• Farr, Christine Jane

• Topoisomerase 2
• TOPBP1
• mitosis
• chromosome compaction
• metaphase congression
• genome stability
• centrosome

Licence

• https://www.repository.cam.ac.uk/bitstreams/aacd9a99-8922-4c58-ad85-e37b7f29a6ce/download
• http://purl.org/NET/rdflicense/allrightsreserved

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.118915

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/385193