For cellular life to exist, genetic material must be copied and passed on to newly-divided cells. In eukaryotes, this process is phenomenally accurate and occurs with an error rate of around one in a billion. The fidelity of DNA replication is ensured by both the biochemical composition of the replication machinery and careful orchestration of the different stages of replication. Decades of study have yielded much information about; replication ‘initiation’, in which macromolecular machines termed ‘replisomes’ are loaded and activated at ‘origins’; and replication ‘elongation’, in which replisomes copy DNA at ‘replication forks’. In contrast, termination, which is the final stage of DNA replication, is poorly understood. In the Dewar lab, we are working to understand the mechanisms that underlie the termination of DNA replication. It is critical for human health that we gain a complete understanding of how DNA replication works, given that various mutations in the vertebrate replication machinery that are sufficient to cause a variety of diseases (from cancer, to drwarfism, and even neurodegeneration) and subversion of DNA replication is an essential part of oncogenesis.
The primary tool used by the Dewar lab is cell extracts, which we derive from eggs of the African clawed frog, Xenopus laevis. These ‘egg extracts’ contain sufficient quantities of cellular proteins to support multiple embryonic cell cycles, even in the absence of translation. Therefore, egg extracts allow us to stimulate chromosome duplication on both full length chromosomes but also customizable plasmid DNAs. To study these processes in unprecedented detail we exploit a plasmid DNA template that contains a reversible replication barrier. This allows us to halt replisomes at a defined location, then synchronously restart them and monitor their progression and composition. This approach recently led to the first biochemical model for replication termination (Dewar et al, 2015, Nature; Dewar et al, 2017, Genes Dev) and also supported studies of DNA repair (Duxin et al, 2014, Cell; Zhang et al, 2015, NSMB). We are currently using this approach to explore replication termination (Heintzman, Campos et al, 2019, Cell Rep) and develop biochemical sytems to study telomere replication.