Functional dissection of the eukaryotic replisome. (360G-Wellcome-102943_Z_13_Z)

We aim to understand the functions of the eukaryotic replisome, which is a critical determinant of genome integrity, and an important target for current and future anti-tumour therapies. One of the greatest challenges faced by eukaryotic cells is to make a single and near-perfect copy of their chromosomes during each cell cycle. As well as unwinding and copying a vast amount of DNA without mistakes or breakage, the cell must also duplicate all the complex patterns of histone modifications that determine epigenetic patterns of gene expression. Moreover, the two sister chromatids produced by replication are held together within large rings of protein called cohesin, in a process that must be established during chromosome replication. For all these reasons, the eukaryote replication machinery is highly complex, involving many more components than its better-understood counterpart in E. coli. The DNA helicase and polymerases at replication forks associate with many other factors to form a dynamic assembly known as the replisome. The eukaryotic replisome is still very enigmatic, as it is assembled in situ and does not exist away from replication forks, it cannot be isolated in an intact form, and it contains many components that are still of unknown function. My group has made a series of important contributions to the emerging view of the eukaryotic replisome (Figure 1). Our work helped to define the nature of the 11-subunit Cdc45-MCM-GINS DNA helicase [1-3], togeth er with its interactions in the replisome with a set of regulatory factors and both leading and lagging strand DNA polymerases [2, 4-7]. The key challenge now is to understand how the complex structure of the eukaryotic replisome underlies unique functions that allow it to preserve genome stability, contribute to the inheritance of epigenetic information, and also fulfil other roles such as helping to establish cohesion between sister chromatids. Though poorly understood in all eukaryotes, the se processes are likely to have been very highly conserved across evolution, as almost all replisome components have a single orthologue in each eukaryotic species. We will address the following important questions: (i) How does the eukaryotic replisome contribute to the preservation of parental chromatin? (ii) How does incorporation of the leading strand DNA polymerase into the eukaryotic replisome control replisome function and the rate of fork progression? (iii) How is the lag ging strand machinery incorporated into the eukaryotic replisome and does this mechanism provide a novel target for drug development?