Herlihy, AE; (2017) The DNA replication stress checkpoint transcriptional response and its role in replication stress tolerance. Doctoral thesis , UCL (University College London).

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Abstract

DNA replication stress is defined as the slowing down or stalling of DNA replication forks. The DNA replication stress checkpoint prevents replication stress from developing into DNA damage and subsequent genome instability. This checkpoint response maintains E2F-dependent cell cycle transcription through inactivation of E2F6. However, the role and importance of transcription in response to replication stress remains largely unknown. Work presented in this thesis shows that in mammalian cells, unlike yeast, active protein synthesis is required for an efficient checkpoint response. Many checkpoint effector proteins, which are E2F targets, are found to be unstable and so require sustained E2F-dependent transcription to maintain levels during replication stress. Maintaining correct protein levels is necessary for specific functions of the checkpoint response - replication fork stalling, stabilisation and protection, and resolving stalled forks after stress. E2F-dependent transcription is therefore required to prevent DNA damage following replication stress. Importantly, maintaining E2F-dependent transcription during replication stress in checkpoint-compromised cells is sufficient to prevent DNA damage. These results suggest that sustained E2F-dependent transcription is a key mechanism in the replication stress response. Activated oncogenes increase E2F activity, driving uncontrolled proliferation, thought to be at the basis of oncogene-induced replication stress, an early event in tumourigenesis. Data presented shows E2F-dependent transcription is also required to prevent DNA damage during oncogene-induced replication stress; suggesting oncogenic cells rely on E2F-dependent transcription for tolerance to high levels of replication stress. Given the important role of E2F-dependent transcription in the replication stress response, the mechanism of E2F6-dependent repression is also investigated. Overall my work establishes that E2F-dependent transcription is a key mechanism in the response to DNA replication stress and is necessary during oncogene-induced replication stress. The regulation and functions of this transcriptional response are elucidated, showing a far greater role than previously suspected in the response to DNA replication stress.

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