Maintenance of genome stability is critical for cell survival, and consequently cells have evolved a complex set of mechanisms to ensure DNA is repaired and correctly replicated before cell division. Deficiencies in these repair pathways are heavily associated with the development of cancer and ageing, and it is therefore of interest to be able to detect and monitor the distribution of DNA damage and replication events across the genome.

This work describes the development and optimisation of TrAEL-seq (Transferase-Activated End Ligation Sequencing), a novel sequencing method for genome-wide detection of DNA double strand breaks, replication fork stalling, and replication fork movement. Our method captures single-stranded 3’ DNA ends, which can be mapped across the genome with base pair resolution. TrAEL-seq requires no labelling, synchronisation or sorting of cells, making it very flexible and simple to implement. In this work I refined the protocol to suit studies of mammalian cell systems; I developed a multiplexing protocol for high-throughput sample processing and comparative quantitation, and optimised themethod’s compatibility with fixed cells.

Through collaboration with Artios Pharma, I then used TrAEL-seq to investigate the effect of different DNA damage response inhibitors in cancer cells. Comparison of DNA replication between different therapies and time points provided interesting insights on the mechanism of action of such treatments. Surprisingly, this TrAEL-seq data did not reveal distinct genomic locations vulnerable to replication fork stalling or double-strand breaks, supporting the notion that such events occur in a more random distribution pattern across the genome.

I then applied TrAEL-seq to study an alternative model of DNA replication stress and damage; oncogene-induced senescence (OIS) resulting from HRAS^G12V overexpression. Analysis of TrAELseq replication fork movement across a time course of OIS revealed global changes in the levels of DNA replication across the genome. Interestingly, the data revealed an accumulation of TrAEL-seq signal around a subset of R-loops in senescent cells, which were localised in subtelomeric regions. Follow-up investigations which characterised the nature and origin of these peak sites offer new perspectives on cellular senescence. Taken together, this work demonstrates TrAEL-seq as a novel and exciting tool to investigate a range of dynamic and complex mammalian cell systems.