Balancing Dissolution and Resolution / Finding a Solution

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 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). Critical in safeguarding chromosomal stability is the multi-functional protein TOPBP1, often highly expressed in cancers and essential to survival, which holds many important protein interactions throughout the cell cycle. Importantly, loss of TOPBP1 leads to dysfunction in cell cycle progression and also the stability of the genome.

Recently, we determined that TOPBP1 holds interactions in dividing cells with the multi-protein complexes the 'BTRR' and 'SMX 'complexes. These protein complexes are associated with the repair of persistent DNA replication and DNA repair intermediates that intertwine sister-chromatids in late stages of the cell cycle, as such these complexes enable the separation of the chromatin between daughter cells and prevent genome damage. As such, the present project aims to characterise the role of TOPBP1 in the regulation of BTRR and SMX functions, at precise stages throughout the cell cycle and also at specific positions within the genome. This is to determine if these interactions are spatially or temporally distinct or local competition is regulated by TOPBP1. To achieve this we will use previously generated cell line models that are defective for interactions between TOPBP1 and SMX or BTRR to aid in delineating their functional role. These cell lines will be examined with use of state of the art microscopy approaches, to precisely track changes in the recruitment of components of the BTRR and SMX complexes and also characterise unique consequences to chromosomal stability at specific stages in the cell cycle and to specific positions in the genome. Further to this, we will explore the signalling mechanisms that regulate the disentanglement of chromatin and also safeguard chromosomal stability.

This highly collaborative project will work with the Chan laboratory to employ high resolution microscopy approaches to visualise DNA entanglements that persist into the final stages of the cell cycle, as so called 'chromatin bridges and with the Pearl laboratory we will employ in vitro biology approaches to aid in the identification of the precise interaction surfaces of key proteins involved in the signalling and regulation of the TOPBP1, SMX and BTRR functions to facilitate disentanglement of the intertwined genome. Continued collaboration with the Choudhary laboratory will aid in the assessment of changes in TOPBP1 protein interactions and signalling events that may be involved in a common mechanism of chromosomal disjunction. To translate the fundamental biological findings of these studies to potential benefit to patients we will also determine if disrupting these processes improves the effectiveness of established clinically relevant anti-cancer therapies.

This innovative research project aims to provide new insight into the key processes that govern cell cycle progression, DNA repair and cell division, providing new scope for the development of novel rationale for the treatment of cancer and other genetic diseases.

The goal of the study is to understand the role of TOPBP1
and its interactions with the BTRR and SMX complexes, in facilitating chromosome disjunction and maintenance of chromosomal stability. Three aims will address this with the aid of a collaborative network:
i) Delineate the mechanisms of spatio-temporal control of the TOPBP1-BTRR- SMX axis: Previously generated cell lines harbouring separation of function mutations that disrupt interaction between TOPBP1 and SLX4 or BLM will be used to explore their functional role in safeguarding genome stability and chromosomal disjunction.
ii) Characterise the functional role of mitotic post-translational regulation of TOPBP1 on its ability to regulate chromosomal disjunction: Previously generated cell lines harbouring deletion mutations of identified mitotic PLK1 docking sites on TOPBP1 will be used to determine their role in cell cycle progression and genome stability.
iii) Examine the impact of disrupting the TOPBP1-SMX-BTRR axis and its regulation on genome stability as well as its potential application in cancer therapy.
High resolution spinning disc imaging and widefield immunofluorescence microscopy approaches will be used to determine the effects of separation of function mutations on the recruitment of components of the SMX/BTRR and genome stability and cell cycle progression. We will assess the spatial and temporal regulation of these processes by classification of cells with loci specific markers and cell cycle markers and evaluate epistasis with recombination and CDK1/PLK1 regulation. We will explore the complexity of the TOPBP1-SMX sub-complex interaction and reconstitute TOPBP1 CDK1/PLK1 phosphorylation in vitro via recombinant protein purification combined with biophysical and structural analysis. Finally, we will explore the sensitivity profile of disrupting the identified mechanisms in combination with established DDR and checkpoint inhibitors measuring cell survival and proliferation phenotypes.