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

Structure and regulation of the human INO80–nucleosome complex

By Rafael AyalaOliver WillhoftRicardo J. AramayoMartin WilkinsonElizabeth A. McCormackLorraine OclooDale B. Wigley & Xiaodong Zhang

Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines1. Biochemical studies2,3,4 have placed the motor domains of several chromatin remodellers in the superhelical location 2 region of the nucleosome. Structural studies of yeast Chd1 and Snf2—a subunit in the complex with the capacity to remodel the structure of chromatin (RSC)—in complex with nucleosomes5,6,7 have provided insights into the basic mechanism of nucleosome sliding performed by these complexes. However, how larger, multi-subunit remodelling complexes such as INO80 interact with nucleosomes and how remodellers carry out functions such as nucleosome sliding8, histone exchange9 and nucleosome spacing10,11,12 remain poorly understood. Although some remodellers work as monomers13, others work as highly cooperative dimers111415. Here we present the structure of the human INO80 chromatin remodeller with a bound nucleosome, which reveals that INO80 interacts with nucleosomes in a previously undescribed manner: the motor domains are located on the DNA at the entry point to the nucleosome, rather than at superhelical location 2. The ARP5–IES6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This arrangement enables the histone H3 tails of the nucleosome to have a role in the regulation of the activities of the INO80 motor domain—unlike in other characterized remodellers, for which H4 tails have been shown to regulate the motor domains.