Before a cell divides it has to replicate its genetic material so that each new daughter cell can receive a copy. This process of DNA replication is performed by complex protein machines, which are assembled onto DNA at chromosomal sites known as replication origins. During S-phase of the cell cycle, multiple origins along each chromosome fire each releasing a pair of replication forks that travel in opposite directions away from their point of initiation. DNA replication is completed when forks from adjacent origins merge. The failure of even a single pair of forks to merge results in a region of unreplicated DNA that can lead to aberrant chromosome segregation during mitosis, DNA breakage and ultimately genetic mutations that cause diseases such as cancer. Replication fork barriers, including DNA lesions, transcription complexes and DNA binding proteins, threaten the successful completion of DNA replication by physically impeding the progress of the replication fork. Elaborate mechanisms enable replication forks to overcome such roadblocks and safely and accurately merge during replication termination. Our goal is to elucidate these mechanisms.
Elucidating the mechanisms that govern replication restart efficiency and fidelity
Replication stress is a major cause of genomic instability that drives tumorigenesis and other pathological states. Exactly what the stress entails, and how this results in genomic instability, is the subject of intense investigation, with altered patterns of replication origin firing, aberrant processing of replication forks stalled at barriers, and genomic instability wrought by recombination-dependent replication thought to be major contributory factors. Despite their importance, our understanding of the molecular basis of these processes is still rudimentary and far from complete. In pioneering work, we have established a unique set of tools to investigate the processing of perturbed replication forks at site-specific barriers in the model eukaryote fission yeast (e.g. Nguyen et al 2015). Using these tools, together with state-of-the-art in vivo biochemistry, advanced microscopy and cutting-edge high-throughput single molecule DNA fibre (HTDF) analysis, we aim to deliver a comprehensive description of the factors that influence the processing of a blocked replication fork through its collapse, restart by recombination, and progression as a restarted fork. In particular we are focusing on identifying the factors that affect the efficiency and fidelity of these processes, and determining whether they are similarly required for processing broken replication forks. Replication fork failure is thought to trigger the firing of nearby dormant replication origins to aid the timely completion of genome duplication. However, it is unclear whether this is an active or passive process, and whether it depends on wide-scale induction of a checkpoint response or can be triggered locally by the collapse of a single fork. Using HTDF analysis, we aim to answer these important questions, establishing the pattern of replication origin firing in response to site-specific collapsed and broken replication forks, and determining whether recombination dysfunction alters the patterns observed both locally and genome-wide.
Inter-Fork Strand Annealing: a novel process that causes genomic deletions during the termination of DNA replication
Problems that arise during DNA replication can cause genomic alterations that are a major driver of ageing and age related diseases such as cancer, as well as being instrumental in the development of many human genetic disorders. Replication fork barriers are one common problem encountered, which can cause the dissociation of replisome components (fork collapse), and act as hotspots for replication termination, where replisomes from adjacent replication origins converge. Collapsed forks can be rescued by homologous recombination, which restarts replication. However, replication restart is a relatively slow process and, therefore, replication termination frequently occurs here by an active fork converging on a collapsed fork. We have discovered this type of non-canonical fork convergence in fission yeast is prone to trigger very high frequencies of deletions between repetitive DNA sequences. We hypothesise that deletions occur during replication termination via a novel mechanism that we call inter-fork strand annealing (IFSA) (Morrow et al 2017). Due to the abundance of repetitive DNA elements in the human genome, we suspect that IFSA presents a major and completely novel threat to human genome stability that likely contributes to ageing and age related diseases. Using a combination of state-of-the-art genetic, live cell imaging and biochemical approaches, we aim to establish the validity of our model, and uncover the key factors that influence it. Understanding the molecular mechanisms responsible for promoting and suppressing IFSA will provide a platform for future studies that could ultimately lead to the development of new approaches for minimising the deleterious effects of ageing, including novel diagnostics, prognostics and therapeutics for diseases such as cancer.
Our work is supported by funding from the BBSRC and MRC
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