Professor Michael O’Donnell – A Twin DNA Replication Factory
For life on Earth to grow, its genetic material must be copied and reproduced in a process known as DNA replication. Professor Michael O’Donnell, head of the Rockefeller University’s DNA replication laboratory, has devoted his over 30-year career to the study of the protein complex that is responsible for just that – the replisome. Recently, Professor O’Donnell and his team uncovered exciting insights into the function of this remarkable piece of molecular machinery.
Behind the Scenes of DNA Replication
From conception, a single fertilised egg cell faces exponential growth in order to become a two trillion cell new-born. During each round of cell division, DNA – copied at a rate of 25 nucleotide units per second – requires extreme accuracy during replication, since any mistake has the potential to be fatal. DNA stores the genetic blueprint of every organism in a series of nucleotide chemical bases known as adenine (A), thymine (T), guanine (G), and cytosine (C). These four letters form a unique genetic code underpinning the characteristics of nearly every life form on Earth.
DNA consists of two complementary, anti-parallel strands that coil around a common axis in the shape of a double helix. A base from one strand pairs with a base from the anti-parallel strand in the following manner: A pairs with T, and G pairs with C. While the order of bases is ever-changing, the manner in which they pair is not. It may seem paradoxical, therefore, that DNA replication occurs so smoothly. Enter the replisome.
‘Every time that a cell divides to form two new cells, the DNA instructions for life must be duplicated in a timely and accurate fashion’, emphasises Professor Michael O’Donnell from the Rockefeller University, describing what has been the focus of his work for over thirty years. ‘This remarkable feat is accomplished by a machinery somewhat like a sewing machine composed of many protein “gears” that function together, referred to as a replisome.’
Professor O’Donnell heads the Rockefeller University’s DNA replication laboratory and has devoted his career to understanding the unique architecture of proteins contained within the replisome, by studying both their physical structures and biochemical activities. His work aims to provide new insights into cellular replication, repair, and genetic inheritance.
From Baker’s Yeast to Humans
Over a billion years of evolution, the components of the replisome have been largely conserved across the animal, plant and fungi kingdoms. One of the leading protein ‘gears’ found within is helicase, responsible for separating the two DNA strands. DNA must be unwound for replication to occur, going from a tightly coiled double helix to two straight lines, much like the unzipping of a zip. From baker’s yeast to humans, the helicase found within the replisome is an 11-protein assembly called CMG, named for its three components: Cdc45 protein, the MCM2-7 motor ring, and GINS complex.
This unwinding is achieved by CMG encircling one strand of a double-stranded DNA molecule via its MCM ring and excluding the other strand to the outside of the ring. Then, CMG tracks along the encircled strand, acting as a moving wedge to split the DNA duplex apart.
CMG’s other two elements – the Cdc45 protein and GINS complex – are used to attract and capture other protein components of the replisome machine. These include DNA polymerases, used to synthesise DNA by assembling individual nucleotides (building blocks) of DNA; a ring-shaped sliding clamp called PCNA, used to tether DNA polymerases to parent strands of DNA; a clamp loader protein that allows PCNA to clamp around DNA; and a primase, called DNA polymerase α-primase (DNA pol α-primase), which synthesises primers required to initiate DNA replication.
‘Every time that a cell divides to form two new cells, the DNA instructions for life must be duplicated in a timely and accurate fashion…This remarkable feat is accomplished by a machinery somewhat like a sewing machine composed of many protein “gears” that function together, referred to as a “replisome”’.
A Factory of Twin Replisomes
DNA replication occurs in the nucleus, or central organelle, of each cell. ‘It has long been known that replication proteins localise to various “spots” in the nucleus that can be observed in the microscope and are referred to as replication foci’, explains Professor O’Donnell. ‘These are the sites of DNA replication. Studies in yeast showed that these spots mainly consist of only two replisomes. Therefore, it would appear that replication occurs in factories of twin replisomes.’
Indeed, Professor O’Donnell and colleagues discovered that a novel player is crucial to the formation of the twin replisome factory: a protein called Ctf4 (Chromosome Transmission Fidelity 4). Ctf4 is a homotrimer, meaning it has three identical subunits, and two of these are used to tightly bind CMG molecules. Nevertheless, the exact structure of this twin CMG-Ctf4 unit remained unknown. This is where Professor O’Donnell teamed up with Dr Huilin Li of the Van Andel Institute in Michigan, an expert in a technique known as cryogenic electron microscopy (cryo-EM) used to examine the high-resolution structures of biomolecules.
With the help of Dr Li, Professor O’Donnell discovered that the mystery CMG-Ctf4 structure was that of two CMG helicases oriented in a head-to-head fashion around one Ctf4 protein. This unique layout explains the observation that DNA foci in yeast contain two replisomes. In humans, however, nuclear foci are bigger but super high-resolution imagery has revealed that each large focus is simply composed of many sub-foci, and each of these sub-foci is a twin replisome. Professor O’Donnell and his team thus uncovered a key finding underpinning the DNA replication factory: twin replisomes are held by a Ctf4 scaffold in a head-to-head fashion.
CMG Unwinds DNA in the N-first Direction
DNA replication is an inherently dynamic process: a helicase must traverse an entire DNA molecule in order to split it into two strands and enable duplication. CMG, like other proteins, has an asymmetric structure, defined by the N- and C-ends of the proteins. The orientation of CMG as it travels on DNA was another mystery that the O’Donnell laboratory was keen to solve. Does it travel N-first or C-first?
The replication factory mechanism occurs as follows: a parental, double-stranded DNA molecule feeds into the system and is separated into two single strands. Of these, one strand threads through CMG, while the other remains outside the CMG ring at the centre of the factory. Professor O’Donnell, with the help of Dr Li, used cryo-EM to enable direct visualisation of this process to investigate CMG’s direction of travel. ‘We determined the orientation of CMG helicase while it travels on DNA, which was opposite from the orientation that had been assumed by the field for over a decade. This “challenge” has been confirmed by other labs now’, explains Professor O’Donnell.
This visualisation showed the fates of each DNA strand. After being threaded through CMG, one DNA strand was duplicated by a polymerase ε protein (DNA pol ε). DNA pol ε, which directly bound CMG, was held in place by the aforementioned PCNA clamp which encircles double-stranded DNA. The duplication of the strand not threaded through CMG, termed the lagging strand, was attended by DNA polymerase δ (DNA pol δ). DNA pol δ replicated the lagging strand thanks to the primers synthesised by DNA pol α-primase while also being held in place by the PCNA sliding clamp.
This cryo-EM visualisation led to another key discovery in solving the replisome puzzle: only one DNA pol α-primase could bind the Ctf4 trimer. Consisting of three subunits, the Ctf4 trimer, therefore, bound two of these to CMG molecules, and the third to DNA pol α-primase, implying that DNA pol α-primase must split its primer forming activity between the two lagging strands. Slowly but surely, Professor O’Donnell was shining a light on the previously hidden intricacies of the replisome.
An Ensured Inheritance
Perhaps surprisingly, every bodily cell contains the entire selection of DNA or genome. However, only certain genes end up being ‘switched on’. Whether or not a gene is expressed depends on epigenetic markers, which, in a nutshell, are chemical tags on DNA. DNA is packaged inside a cell into functional units called nucleosomes which include these markers, ready to determine the function of each cell. Thus, epigenetic inheritance is an essential component of cell division which must be preserved by the replisome. In the model described by Professor O’Donnell, two CMGs bind to one nucleosome, suggesting that the twin replication factory itself facilitates the transfer of nucleosomes to new DNA, ensuring developmental epigenetic inheritance is achieved.
Professor O’Donnell and his team also proposed that the replication factory helps to organise the newly synthesised DNA genome and that the twin model is able to communicate with itself. Professor O’Donnell summarised that ‘it is possible the two replisomes communicate their status to one another, such that if one replisome stops due to DNA damage, the other replisome may stop as well’. This communication could be integral for the production of healthy daughter cells, void of rogue DNA damage that could lead to cancer.
Looking to the Future
Professor O’Donnell’s laboratory is looking to validate the conclusions drawn here, both in live mammalian cell studies and in bacteria. Presumably, the twin replication factory is applicable to all life forms and provides key information for furthering the study of DNA replication. The enthusiasm that Professor O’Donnell shares for unlocking new secrets of this field is contagious, as he declares ‘the current study is only the beginning of a comprehensive understanding of how replication is organised in the cell. We expect that additional proteins, further layers of organisation, and yet to be determined dynamic actions of these proteins, exist in nuclear replication factories.’
Meet the researcher
Professor Michael O’Donnell
Department of DNA Replication
The Rockefeller University
New York, NY
Professor Michael O’Donnell received his PhD in biochemistry from the University of Michigan in 1982 and went on to complete a postdoctoral position at Stanford University in 1986. At the close of his postdoctoral studies in 1986 he founded his research group at Cornell Medical College in New York City, which he moved to the Rockefeller University in 1996 where he now heads the Laboratory of DNA Replication. Over his 30-year career, he has achieved an impressive accolade of honours and awards including becoming an investigator with the Howard Hughes Medical Institute in 1990 to the present time, and induction into the United States National Academy of Sciences in 2006. Alongside this, he is an editor and reviewer for several research journals and fundraising bodies. His research focuses on mechanistically understanding how the collection of proteins involved in DNA replication ensures genomic integrity. During the COVID-19 outbreak, Professor O’Donnell and his team are investigating potential vulnerabilities in the reproduction of coronavirus.
Dr Huilin Li, Van Andel Institute, Grand Rapids, Michigan
Dr John Kuriyan, University of California at Berkeley, Berkeley, California
National Institute of Health General Medical Institute
Howard Hughes Medical Institute
Z Yuan, R Georgescu, R Santos, D Zhang, L Bai, G Zhao, M O’Donnell, H Li, Ctf4 organizes sister replisomes and Pol α into a replication factory, eLife, 2019, 8, e47405.
R Georgescu, Z Yuan, L Bai, R Santos, J Sun, D Zhang, O Yurieva, H Li, M O’Donnell, Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation, Proceedings of the National Academy of Sciences, 2017, 114, 5, E697–E706.
Z Yuan, L Bai, J Sun, R Georgescu, J Liu, M O’Donnell, H Li, Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation, Nature Structural & Molecular Biology, 2016, 23, 3, 217–224.
Want to republish our articles?
We encourage all formats of sharing and republishing of our articles. Whether you want to host on your website, publication or blog, we welcome this. Find out more
Creative Commons Licence
(CC BY 4.0)
This work is licensed under a Creative Commons Attribution 4.0 International License.
What does this mean?
Share: You can copy and redistribute the material in any medium or format
Adapt: You can change, and build upon the material for any purpose, even commercially.
Credit: You must give appropriate credit, provide a link to the license, and indicate if changes were made.
More articles you may like
Professor David Magnuson, at the University of Louisville, Kentucky, describes himself as ‘a CPG guy’ and occasionally, more informally as ‘a rat guy!’ His work on the function of the central pattern generator (CPG) in the rat spinal cord following spinal cord injury, has produced both surprising and thought-provoking results. This research may ultimately challenge the established clinical beliefs and practices around the ways to best rehabilitate human patients with severe spinal cord injury.
Professor Kim Dale | Dr Hedda Meijer – The Role of Notch Signalling within the Molecular Clock in the Early Development of the Skeleton
Cells possess the ability to interact with one another through complex signalling pathways. Different signals regulate how cells differentiate, undergoing modifications that ultimately allow them to adopt different cell fates and perform specific functions. The laboratory of Professor Kim Dale from the University of Dundee, Scotland, has made seminal contributions to our understanding of how the Notch signalling pathway controls the formation of tissues and organs in the earliest stages of development. Their important research has unveiled new insights into the molecular basis of Notch signalling in the context of normal development which will further our understanding of the molecular basis of developmental disorders and a multitude of diseases correlated with aberrant Notch signalling.
Pancreatic ductal adenocarcinoma (PDA) is an aggressive type of cancer. It is relatively common and is one of the leading causes of cancer mortality. Unfortunately, it is often detected only in the late stage of the disease and fails to respond to pre-surgical approaches, such as chemotherapy or radiotherapy, that are needed to shrink the tumour mass before surgical removal. Dr Scott Gerber at the University of Rochester Medical Center, USA, is working with colleagues to develop a novel combined therapy to overcome this issue and increase the survival of PDA patients.
Dr Elizabeth A. Cooper – New Sorghum Reference Genome Highlights Genetics Underlying Sweet Varieties
Sorghum is a staple crop in many regions of the world. As such, this versatile plant has been selectively bred into a number of cultivars, including sweet varieties predominantly used for forage, silage, sweet syrup and bioenergy production. Dr Elizabeth A. Cooper and her team at the University of North Carolina at Charlotte generated a full reference genome for the sweet sorghum cultivar ‘Rio’ with the aim of understanding the genetics underlying the differences between grain and sweet cultivars. Their research could provide a vital tool for biologists and breeders to improve sweet sorghum lineages.