Credit: Mattiroli group, copyright Hubrecht Institute. 27 March 2023 ‘DNA replication in a tube’ provides new insights into chromatin assembly Back to news Researchers from the Mattiroli group re-created DNA replication and chromatin assembly in a test tube. They discovered that the crosstalk between these two fundamental processes differs between the two newly replicated DNA strands. The results, published on 21 March 2023 in Nucleic Acids Research, can contribute to a better understanding of deregulated chromatin replication in cancer. The cells in our body regularly divide to produce new cells. Before a cell divides, it needs to duplicate its DNA to ensure that the new cells will contain the same genetic information. This occurs during the process of DNA replication. In addition, the cell needs to replicate the packaging system of DNA, called chromatinA name for both the proteins and the DNA in a chromosome.. The chromatin packaging system enables two meters of DNA to fit into the cell nucleus and consists of DNA wrapped around proteins named histonesProteins in the nucleus around which the DNA is wound. This way, all the DNA fits inside the nucleus. Through modifications of the histones, the DNA is wound tighter or looser, which changes the accessibility of the genes in that area.. Apart from its role in tightly packing the DNA, chromatin regulates which parts of the DNA are active in the cell, thereby determining how the cell behaves. As the cell divides, it is therefore essential that not only the DNA code is faithfully replicated, but the chromatin information as well. Otherwise, the newly formed cells will start to malfunction, which can lead to diseases such as cancer. Taking chromatin replication out of the cell While DNA replication has been studied extensively, chromatin replication is much more challenging to investigate. This is due to the complexity of chromatin structure and its many interconnected functions in cells. As a consequence, the mechanisms behind this process remain largely unknown. In order to change this, the researchers used a variety of biochemical systems to re-create the DNA replication and chromatin assembly of yeast in a test tube. Co-first author Clément Rouillon explains: “Within cells, there are many back-up mechanisms and checkpoints to ensure that essential processes like chromatin replication are carried out correctly before the cell divides. This allows the cell to correct errors in these processes, but it can mask the function of individual molecules. Therefore, we took the process of chromatin assembly outside the complex cellular environment. For this we combined new in vitroLiterally: in glass. In vitro research means that a process of the body is studied outside of the body. For example, cells can be used for these studies. chromatin assembly assays with powerful DNA replication approaches previously developed by the Diffley and Yeeles laboratories in the UK. This provides a highly controlled system which allows us to carefully study the process of chromatin assembly, and its link to the large DNA replication machinery which includes over 50 different proteins.” Asymmetrical crosstalk By using these systems, the researchers discovered that chromatin assembly occurs in a different manner on each of the two replicated DNA strands. Co-first author Bruna Eckhardt explains: “During DNA replication, the two strands of DNA can be distinguished as the leading and lagging strand. The leading strand is made continuously, meaning that the DNA is replicated in one piece. The lagging strand is produced in small pieces that are connected afterwards. We already knew that the leading and lagging strand contain different proteins. What we discovered now is that this leads to differences in the way the chromatin machinery affects DNA replication.” The team specifically focused on the protein CAF-1. This so-called ‘histone chaperone’ binds histones to deliver them to newly formed DNA, thereby forming the chromatin packaging system. Eckhardt: “Together with our collaborators in the laboratory of Anja Groth at the University of Copenhagen, we show that CAF-1 indeed makes chromatin on both DNA strands. However, the interplay between CAF-1 and the enzymesA protein that accelerates a specific chemical reaction, for example the breakdown of a molecule. The enzyme itself remains intact in such a reaction, which enables the molecule to exert its function over and over again. Enzymes are present both in- and outside the cell. involved in DNA replication differs between the two strands.” Rouillon adds: “Our results suggest that DNA replication and chromatin assembly cannot occur simultaneously on the leading strand, while the two processes might be coupled on the lagging strand. This is consistent with the fact that DNA replication is in itself an asymmetrical process, with differences between the two DNA strands.” Visualization of the amount of DNA produced by the leading strand DNA polymerase, the enzyme responsible for DNA replication (pink bands). From left to right, increasing amounts of CAF-1 were added, leading to stronger inhibition of DNA replication (diminishing band intensity). This suggests that chromatin assembly and DNA replication cannot occur simultaneously on the leading strand. This effect was not found on the lagging strand. Credit: Mattiroli group, copyright Hubrecht Institute. Chromatin assembly as a therapeutic target The results of the study are surprising, because the processes of DNA replication and chromatin assembly were thought to occur independently before. “Our data highlight that there is in fact a functional interaction between the two, meaning that affecting one will influence the other,” says group leader Francesca Mattiroli. Not only do these results shed new light on DNA and chromatin replication, fundamental processes which we cannot live without, they also give new directions for cancer research. Mattiroli: “These processes are often deregulated in cancer cells, and we know that CAF-1 is involved in the progression of several types of cancer. Therefore, understanding the normal function of CAF-1 will bring us closer to developing new cancer treatments that target this protein.” Publication CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands. Clément Rouillon*, Bruna V. Eckhardt*, Leonie Kollenstart*, Fabian Gruss, Alexander E.E. Verkennis, Inge Rondeel, Peter H.L. Krijger, Giulia Ricci, Alva Biran, Theo van Laar, Charlotte M. Delvaux de Fenffe, Georgiana Luppens, Pascal Albanese, Koichi Sato, Richard A. Scheltema, Wouter de Laat, Puck Knipscheer, Nynke H. Dekker, Anja Groth and Francesca Mattiroli. Nucleic Acids Research, 2023. * Authors contributed equally Clément Rouillon, Bruna Eckhardt and Francesca Mattiroli. Francesca Mattiroli is group leader at the Hubrecht Institute.