Of the many proteins that are required to duplicate a chromosome, a subset associate with the DNA helicase at replication forks to form an enigmatic molecular machine known as the replisome (e.g. see Kanemaki et al, Nature, 2003; Gambus et al, Nat. Cell Biol., 2006, Gambus et al, EMBO J., 2009, Sengupta et al, Curr. Biol., 2013). Assembly of the replisome connects the helicase to DNA polymerases, thus coupling the rate of the DNA unwinding to the rate of DNA synthesis. However, the eukaryotic replisome also contains many factors that are still of unknown biochemical function (Gambus et al, Nat. Cell Biol., 2006), which are not present in the much simpler replisomes of bacteria. This reflects the fact that chromosome duplication in eukaryotes is not just about making two new strands of DNA, but also involves other fascinating but poorly understood processes, such as the regeneration of chromatin and of epigenetic information, the preservation of genome integrity, and the establishment of sister-chromatid cohesion.
We found that budding yeast has unique advantages for the isolation of replisome complexes from replication forks (Gambus et al, Nat. Cell Biol., 2006, Gambus et al, EMBO J., 2009, Sengupta et al, Curr. Biol., 2013), and our goal has been to define the nature of the eukaryotic replisome, and then explore its structure, functions and regulation. In particular, we want to understand how replisome assembly contributes to the unique features of chromosome duplication in eukaryotes described above. As part of this work, we have collaborated with the structural biologists Luca Pellegrini and Alessandro Costa to study the Ctf4 protein, which we found to link the CMG helicase (Gambus et al, Nat. Cell Biol., 2006) to DNA polymerase alpha that initiates Okazaki fragments on the lagging strand (Gambus et al, EMBO J., 2009). Interestingly, Ctf4 forms a trimer (Simon et al, Nature, 2014), and Fabrizio Villa has shown that trimeric Ctf4 serves as a hub in the eukaryotic replisome (Villa et al, Mol. Cell, 2016), linking the CMG helicase to a range of factors with diverse roles in chromosome duplication. Many interesting questions still remain unanswered!