Every time a cell divides, it must copy its entire genome—and in cancer cells, this process is constantly under threat of going wrong. The protein TIMELESS helps protect the DNA copying machinery from this damage, and the researchers want to understand exactly how it does that, both with and without its partner PARP1. This matters because drugs that block PARP1 are already used to treat certain cancers, but doctors do not fully understand why they work. The current evidence suggests PARP1 inhibitors kill cancer cells by disrupting how replication forks handle stress or by creating gaps in newly copied DNA. TIMELESS appears to be involved in both processes, yet its precise role remains unclear. Without this knowledge, it is hard to improve existing treatments or predict which patients will benefit. If the project succeeds, it will map the molecular pathways that TIMELESS uses to manage replication stress. This could reveal why PARP1 inhibitors are effective only in some cancers and suggest ways to make them work in more patients. The research is fundamental science—it does not aim to produce a new drug tomorrow. But understanding how cells protect their DNA during replication has historically led to breakthroughs in cancer therapy, and this work targets a key piece of that puzzle.
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Disruption of the process of DNA replication – often termed replication stress – is a primary source of genome instability in cells. Numerous cellular factors are required to protect cells from the wide range of internal and external sources of replication stress. Cancerous cells develop high levels of replication stress during their transformation from normal tissues. Therefore, cancers become dependent on functions that protect cells from the consequences of replication stress. The continued activity of such pathways allows cancerous cells to tolerate high levels of genome instability whilst dividing rapidly. Specifically targeting replication stress protecting pathways is proving an effective therapeutic strategy in the clinic. Two distinct factors that protect cells from replication stress are the evolutionarily conserved replisome protein Timeless (TIM) and the DNA repair protein PARP1. TIM is a highly evolutionarily conserved part of the DNA replication machine, where it acts to regulate and protect replication fork functions. TIM is also highly expressed in cancer cell lines where it is required for rapid cell multiplication. Parylation of numerous proteins by the enzyme PARP1 is a key feature of single strand break repair and protecting replication forks disrupted by replication stress. While PARP1 interacts with several DNA repair proteins in cells to aid DNA break repair, it is also directly associated with the replication machinery through an interaction with the PAB (PARP1 Binding) domain of TIM. This interaction suggests that the replication stress protection functions of TIM and PARP1 are dependent on one another. Inhibitors of PARP1 (PARPi) have proved to be effective therapies for cancers with defective homologous recombination (HR) repair. Understanding how PARPi specifically kill HR defective cells is crucial for designing therapies that maximise their clinical use. This is a highly active area of research and current results argue that PARPi either target replication forks stalled by replication stress or cause the accumulation of single stranded DNA gaps following DNA replication. Interestingly, several studies have reported that the PAB domain of TIM is also important at stalled replication forks and for single stranded DNA gaps. This argues that the PAB dependent TIM-PARP1 interaction is a crucial regulator of the effects of PARPi. Our groups have extensively researched the roles of TIM and PARP1 in DNA replication. We have recently examined the roles of TIM, the PAB domain of TIM, PARP1 and the TIM-PARP1 interaction during DNA replication and following DNA replication stress. We have found evidence that the PAB domain of TIM has novel PARP1 independent as well as PARP1 dependent roles in the accumulation of single stranded DNA gaps behind the replication fork and following replication fork stalling. To test our new models of action for TIM and PARP1 functions during replication we will Examine how the PARP1 dependent and independent functions of TIM regulate the accumulation of single stranded DNA gaps during DNA replication. Examine how the PARP1 dependent and independent functions of TIM regulate replication fork stalling in response to replication stress. Conduct proteomic and genetic interaction screens to determine other factors involved in the PARP1 dependent and independent functions of TIM to protect cells from replication stress. These experiments will allow us to fully describe new pathways of how TIM and PARP1 function during replication stress and their connections to PARPi cell killing in HR deficient cells.
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