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Investigating eukaryotic replisome dynamics at the single molecule level

Periodic Reporting for period 1 - SMI REP (Investigating eukaryotic replisome dynamics at the single molecule level)

Reporting period: 2015-08-01 to 2017-07-31

Our bodies are made of trillions of cells, each containing the instructions for life, DNA. The genetic information in DNA consists of 6 billion nucleobases, like letters in a book, wound together as a 2 metre long double helix. This fits inside cells 250 times smaller than a pinhead and has 70,000 nucleobases copied every second. The nucleobase letters of DNA are arranged like the zip on a coat. Every time a cell is copied so are all the bases; without mistakes. We know that a zipper, or molecular machine, unzips the DNA but, surprisingly, we have no idea how it works. This is what we researched during the project; how does the zipper work? The problem is that the zipper is 10,000 times smaller than the width of a human hair.

To study the DNA zipper, or helicase, most biologists perform average measurements but the intricate dynamic details of DNA replication cannot be investigated in this way. Instead we wished to observe the processes in real-time, one molecule at a time, and with high spatial and temporal precision. It was therefore necessary to employ a physicist’s perspective and single-molecule biophysics approaches. To this end we have built a single-molecule magnetic tweezers to interrogate 100s of individual DNA molecules simultaneously and observe the action of single helicases unwinding DNA in real-time. We are able to remove ensemble averaging, observe dynamics and discover heterogeneities; enabling quantitative biophysical models to be built that accurately and precisely describe how protein complexes perform their function.

Using the tools and analysis framework built as part of this project we have established that the DNA zipper found in animals like ourselves does not behave as expected. It was assumed the helicase would work in a very coordinated, repetitive manner, like walking. However, we have discovered that to unwind DNA the helicase moves randomly along the DNA, wiggling back and forth due to thermal noise and only slowly moves forward to unwind DNA. In fact, at times, it is more likely to be paused doing nothing.

This is a surprising finding given the central role this helicase has in DNA replication of mammalian cells. If the process of copying DNA is not performed perfectly then the stability of the genome can be impacted, causing DNA damage and hence disease, such as cancer. The understanding we have reached regarding the core process of all cell division in our bodies, unwinding of DNA, is crucial to future human health.
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 @DanBurnham, speaking at local secondary schools, lecturing at scientific societies, attending parliament and organising and speaking at ‘Pint of Science’.
The research carried out has increased the understanding of eukaryotic DNA replication at a fundamental level. The work performed will not immediately have commercial promise but it will provide the building blocks of future medicines and treatments. For example, it is known certain anti-viral and anti-microbial drugs inhibit replicative helicases. Using the technology developed here it will be possible to understand their mechanism of action in greater detail in order to improve the efficacy.
The single-molecule magnetic tweezers microscope constructed as part of this project.