Researchers can now visualise hotly debated paradox of DNA replication

Breaking new ground 20. dec 2022 3 min Group Leader, Postdoc Hana Polasek-Sedlackova Written by Kristian Sjøgren

Minichromosome maintenance proteins (MCMs) have long been a mystery for researchers. Two years ago, researchers discovered why cells have excess inactive MCMs in connection with DNA replication. Now they can also see the proteins. A researcher involved in the study says that the discoveries are important for the fundamental understanding of cell proliferation, and the technique used can be harnessed in studying many other proteins.

During cell division, cells launch extremely complex machinery involving several thousand proteins to duplicate the entire DNA. MCMs (MCM2-7) are essential in this machinery and play a critical role in the cell cycle. They bind to the DNA and assist in synthesising DNA during replication.

However, MCMs have puzzled researchers for many years because they have not understood why so many MCMs need to be used in cell replication and why the proteins have not been able to be visualised in action during replication.

Researchers solved the first paradox in 2000, and now the same research group has finally developed a method that enables MCMs to be visualised in action when one genome becomes two.

“We could not see the proteins with the methods normally used to study proteins in cells and could not understand why not. We have now solved both parts of the paradox,” explains Hana Polasek-Sedlackova, Associate Professor, Department of Cell Biology and Epigenetics, Institute of Biophysics, Czech Academy of Sciences, Brno, Czechia and Novo Nordisk Foundation Center for Protein Research, University of Copenhagen.

The research has been published in Nature Communications.

Inability to see proteins in living cells

The problem with MCM proteins was to visualise them at the sites of DNA synthesis inside a cell.

For many years, researchers extracted MCM proteins from the cells and studied them in a test tube, but to really understand the function of the proteins, researchers need to see them in action during cell replication. When researchers study proteins in cells, they normally use a method to immunostain the proteins so they can see them under a microscope, including colour-coded antibodies.

The problem with MCMs, however, has been that a conventional method of staining proteins with antibodies did not work, and thus it was not possible to verify that the proteins have the predicted function during replication..

This has led some scientists to doubt that they even play the role attributed to them. The researchers behind the new study wanted to change this.

“Our study aimed to visualise MCMs inside a cell and thus provide the definitive proof of their function,” says Hana Polasek-Sedlackova.

Markers tagged to proteins using CRISPR technology

Instead of using traditional methods to stain proteins in cells with antibodies, the researchers used CRISPR-Cas9 genome editing to attach a fluorescent molecule to MCMs in living cells.

They examined the cells under the microscope and very clearly saw the MCMs in the replisome structure that copies the DNA molecule into two.

Hana Polasek-Sedlackova says that the interactions between MCMs and replisomes were exactly as the researchers had described based on experiments in test tubes and that the visualisation thus provided definitive proof for the hypothesised function of MCMs.

(Im)possible to immunostain proteins

In the next part of the research, Hana Polasek-Sedlackova collaborated with structural biologists, who are experts in understanding how protein scaffolds determine the function of proteins.

The researchers hypothesised that the antibodies could not stain the MCMs because some of the proteins that make up the replisome block MCM antibody binding sites.

In one experiment, the researchers removed one of these proteins and found that this made the antibody binding site accessible and thus enabled the MCMs to be stained with antibodies.

“After we removed one protein and were able to stain the MCM proteins with antibodies, we found the same result as in our experiments in which we labelled the proteins with CRISPR technology. This confirmed our findings and finally solved the paradox surrounding the MCM proteins,” explains Hana Polasek-Sedlackova.

Can advance researchers’ knowledge on human health

Hana Polasek-Sedlackova says that the study indicates the importance of studying cellular processes using various techniques, because some methods can identify things that others cannot.

Hana therefore also envisions that the CRISPR protein labelling technology can be used to visualize other proteins that cannot be stained with the current antibody technologies, and this may help to answer unanswered questions about protein function.

In this study, the researchers learned more about cell replication as a fundamental process for life, and studying what happens when one cell becomes two will now be easier. However, Hana Polasek-Sedlackova also thinks that the technology can answer more clinically relevant questions.

“Understanding the role of the molecular scaffold for human health and understanding what goes wrong when we get ill is important. This scaffold cannot be studied only in a test tube; we also need to study MCMs in living cells, and this can be done more easily with the method we used here,” she concludes.

In 2016, she joined the research group of prof. Jiri Lukas at Novo Nordisk Foundation Center for Protein Research as a Ph.D. student within the Copenh...

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