Bacteriophage and prokaryotic studies of DNA replication at the single molecule level revealed monotonic, homogenous and linear dynamics, and it was presumed this would be the case when moving into the eukaryotic system. However, surprisingly, it was discovered that even the core process at the heart of the replisome, DNA unwinding, was heterogeneous and non-monotonic.
In eukaryotes, the replisome is centred around a core hetero-hexameric AAA+ helicase, Mcm2-7. The helicase is activated at origin firing through recruitment of Cdc45 and GINS, forming Cdc45/Mcm2-7/Cdc45 (CMG). Many details of the operation of CMG are hotly debated, including the orientation of the helicase and geometry of the leading and lagging strands.
Bulk biochemical and structural studies of hexameric helicases, including CMG, speculate that CMG unwinds DNA by a variety of mechanisms. For example, via cyclic DNA translocation where DNA binding hairpins sequentially ‘walk’ along the DNA substrate, or as a ‘pump-jack’ where the N and C terminal hexameric tiers open and close like a clamshell.
We have successfully reconstituted eukaryotic DNA unwinding by the core replicative helicase using the reconstituted holoenzyme CMG from recombinant Drosophila melanogaster recombinant proteins and measured the real-time dynamics of purified Drosophila melanogaster CMG unwinding DNA at the single-molecule level with magnetic tweezers.
We have discovered the eukaryotic replisome is far more dynamic than previously thought. The core replicative helicase of the replisome, considered to be a unidirectional molecular motor, undergoes surprising molecular gymnastics to achieve DNA unwinding. The eukaryotic replicative helicase exhibits a biased random walk and, surprisingly, is more likely to be paused rather than actively unwinding. We can explain why we see relatively slow rates of DNA unwinding in vitro compared to replication fork rates in vivo, which is not because the helicase is slow at unwinding but rather because it spends a significant amount of time paused, not performing any productive motion. This understanding has been brought about by the development of not only a new biological assay but a detailed framework of mathematical and statistical analysis that provides a foundation on which to build our continuing understanding of the eukaryotic replisome.
The research has been disseminated by various formal means including via, speaking at colloquia and retreats, and poster and oral presentations at international scientific meetings. The research has also been disseminated via outreach, public and policy engagement. These include and active online presence on Twitter (@danburnham) and blog at Medium.com (@DanBurnham), speaking at local secondary schools, lecturing at scientific societies, attending parliament and organising and speaking at ‘Pint of Science’.
