SMI REP · Investigating eukaryotic replisome dynamics at the single molecule level

Investigating eukaryotic replisome dynamics at the single molecule level

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.

Data: CORDIS, © European Union