Balancing Dissolution and Resolution / Finding a Solution

在每一次细胞分裂过程中，遗传密码必须在两个子细胞之间精确地复制和分离。基因组的忠实分裂对于防止细胞分裂过程中的损伤至关重要。控制这一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（如人类）中，这些过程的准确性更加迫切，因为人类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对生物体致命。尽管如此，我们目前对解开和分离重复基因组的基本过程的理解仍然很差。不能分解纠缠DNA的细胞会形成“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）。保护染色体稳定性的关键是多功能蛋白TOPBP1，通常在癌症中高度表达，对生存至关重要，它在整个细胞周期中具有许多重要的蛋白质相互作用。重要的是，TOPBP1的缺失会导致细胞周期进程和基因组稳定性的功能障碍。最近，我们确定TOPBP1在分裂细胞中与多蛋白复合物“BTRR”和“SMX”复合物相互作用。这些蛋白质复合物与持续DNA复制的修复和在细胞周期后期缠绕姐妹染色单体的DNA修复中间体有关，因为这些复合物使子细胞之间的染色质分离并防止基因组损伤。因此，本项目旨在描述TOPBP1在整个细胞周期的精确阶段以及基因组中的特定位置对BTRR和SMX功能的调节中的作用。这是为了确定这些相互作用是否在空间或时间上是不同的，或者局部竞争是由TOPBP1调节的。为了实现这一目标，我们将使用先前生成的细胞系模型，这些模型在TOPBP1与SMX或BTRR之间的相互作用中存在缺陷，以帮助描述它们的功能作用。这些细胞系将使用最先进的显微镜方法进行检查，以精确跟踪BTRR和SMX复合物组分招募的变化，并描述细胞周期特定阶段和基因组特定位置对染色体稳定性的独特影响。除此之外，我们将探索调节染色质解开和维护染色体稳定性的信号机制。这个高度合作的项目将与Chan实验室合作，采用高分辨率显微镜方法来观察持续到细胞周期最后阶段的DNA缠结，即所谓的“染色质桥”。与Pearl实验室合作，我们将采用体外生物学方法来帮助识别参与TOPBP1信号传导和调节的关键蛋白质的精确相互作用表面。SMX和BTRR的作用是促进缠绕在一起的基因组的解开。与Choudhary实验室的持续合作将有助于评估TOPBP1蛋白相互作用和信号事件的变化，这些变化可能涉及染色体分离的共同机制。为了将这些研究的基础生物学发现转化为对患者的潜在益处，我们还将确定破坏这些过程是否会提高已建立的临床相关抗癌疗法的有效性。这个创新的研究项目旨在为控制细胞周期进程、DNA修复和细胞分裂的关键过程提供新的见解，为癌症和其他遗传疾病的治疗提供新的理论基础。

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.