DNA replication under the (electron) microscope

Article by Nadezhda Aleksandrova, MRes student at Imperial College London

Dr Alessandro Costa, a group leader at The Francis Crick Institute and an Imperial College alumnus, was the guest lecturer for the Imperial College Section of Structural Biology seminar in April. He presented his work, which is focused on understanding the molecular mechanisms underlying DNA replication in complex organisms such as animals and plants. DNA replication is a fundamental process that allows the entire DNA from a single cell to be copied and divided between two daughter cells when cell division occurs. The mechanism of replication is tightly regulated, as errors arising from copying the genetic code may result in accumulation of mutations within DNA and can subsequently drive cancer development.

 The main technique that Dr Costa’s lab uses to study the mechanisms of DNA replication is single particle- electron microscopy- a cutting-edge technique that allows scientists to visualise single proteins and protein assemblies in very high resolution, down to their building blocks, the amino acid residues. Researchers in Costa’s lab use the technique to model the small movements of the proteins responsible for DNA replication to understand what happens when a DNA strand binds to those proteins and how the process is facilitated by the energy currency of the cell, ATP.

 Initially, Dr Costa and his team were interested in helicase, the enzyme responsible for ‘opening up’ the DNA helix, and how it binds to a strand of DNA. This step is necessary, as the DNA is tightly wound into a double helix that cannot be copied without its structure being partially disrupted to allow for the necessary protein machinery to access the genetic code. The 3D images generated by the scientists at the Crick, shown in the figure below, show that helicase binding alone is not sufficient to separate the DNA strands from one another.

Subsequent research in Costa’s lab reveals that binding of ‘firing factors’ to helicases activates the proteins, triggering DNA unwinding. This occurs through ‘stretching’ of the DNA double helix by the active helicase, resulting in single-stranded DNA regions ready for duplication.

 In addition, Dr Costa’s group has generated a 3D model of how the long stretches of DNA move through the channel of the helicase. This allows the full length of the double helix to be sequentially opened up and copied rapidly and efficiently. Efficiency is key in this process, as cell division occurs in the matter of only an hour, during which a huge stretch of DNA must be copied without any mistakes.

 Apart from the work presented in the seminar, Dr Alessandro Costa is also involved in research on the replisome- the protein assembly that has the job of copying DNA. They also strive to improve sample preparation and analysis of electron microscopy images, as the quality of the sample and the image processing are fundamental for the quality of the resulting 3D structures. 


References: Cryo-EM structure of a licensed DNA replication origin, The mechanism of eukaryotic CMG helicase activation