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Detection of Chromatin Bridges during Cytokinesis

Periodic Reporting for period 1 - NoCut (Detection of Chromatin Bridges during Cytokinesis)

Reporting period: 2016-03-01 to 2018-02-28

Conservation of genome integrity by the NoCut pathway
In order to develop and repair all of the body tissues that living organisms are made from, each of the cells that make up the organism must be copied and divide into two new daughter cells many times. The genetic information contained within these cells is packaged chromosomes and these must also be very carefully coped during this life cycle. This is a very complicated process and requires a set of stringent quality controls that ensure that all genetic information is copied and distributed properly between the two daughter cells each time. These quality controls check that the replication of the genetic material, the DNA, is complete, that is it undamaged and finally that the two daughter copies of the chromosome are properly packaged and separated into the two daughter cells. This system is called the cell cycle and is essential for life.
We are interested in a final quality control, which checks that the replicated chromosomes are properly segregated into the two daughter cells before the daughter cells are permanently separated. This quality control point is called the NoCut Checkpoint and is an important control point as sometimes the migration of the two sister chromosomes to opposite daughter cells is incomplete or fails entirely. If the cell makes the final division once this mistake has been made, genetic information can be physically lost, damaged or mutated. It is very important for the organism that this does not happen as this type of damage can cause death of the individual cell and is also thought to be one of the contributing factors in development of cancer.
The aim of this project was to understand how the cell detects whether or not an error in segregation of the genetic information has been made, and therefore whether to activate the NoCut checkpoint. This is important because it may help us to understand how this mechanism is triggered and therefore give insights into if and why is may fail during cancer progression. This may therefore lead to clues about which parts of the process, if any it would be useful to target in future treatment strategies.
In order to achieve this we used yeast as a model system. Yeast is a very useful research tool as it allows us to easily and quickly make genetic changes to test hypotheses. We can then easily confirm the observations made in yeast cells in human cells to check that they are relevant to human cell biology. During this project, we have now identified 2 genes that are essential for the NoCut checkpoint to function. These genes code for proteins that physically bind to DNA and are able to detect changes in DNA structure due to damage and mistakes in DNA replication. We therefore think that these proteins could be the key to understanding the connection between mistakes in the replication and segregation of genetic material and the NoCut checkpoint. We have also confirmed that a highly related protein performs the same function in human cancer cells giving us a really important insight into how the cell is able to perform this type of error checking and therefore how this may function in cancer cells. The next step in this work will be to further map which other genes interact with this pathway and to use real patient cancer sample to test whether this pathway is functional in cancer cells.
In order to identify which genes were involved in the NoCut checkpoint, we used the yeast system as a genetic tool. In this system we were able to delete individual genes and test the effect of these changes on the way the NoCut checkpoint functions. We deleted genes that are already known to bind to DNA as we were looking for a DNA sensor. We were successful in identifying one such gene, which is a DNA helicase. Deletion of this gene stopped the NoCut checkpoint from functioning entirely suggesting that this is central to the pathway. We unraveled the pathway further by identifying a protein that physically binds to the helicase and we have showed that it is this physical interaction that is important for the NoCut pathway to function. Our main evidence for this was obtained by using timelapse microscopy to make movies of yeast cells with and without problems in DNA segregation. In a normal situation, when a cell encounters a problem in DNA segregation, it triggers the NoCut pathway and this causes a delay in cell separation. In the experimental situations where we have deleted parts, or all of the helicase gene, the cell does not activate this delay and the cell divides despite the mistake in segregation. We have also confirmed this finding by deleting a similar gene in the human HeLa cell line and have preliminary evidence that a related pathway exists in human cells. We have presented these findings in the following scientific meetings:
Barcelona Genetic Instability Meeting (GINBAR) 2018: Oral presentation
Chromosome Dynamics Meeting (Chromodyst) 2018: Oral presentation
Gordon Research Conference 2017: Poster presentation
CRG Postdoc Symposium 2017: Faculty Pick oral presentation

We are also currently preparing a manuscript detailing these finding for submission to a peer reviewed journal in May 2018.
This project has revealed for the first time a protein that can physically detect that a certain type of segregation error has been made during the cell cycle. This will now allow us to investigate whether this pathway is relevant in human cancers. The expectation is that that pathway is highly active in cancer cells with genomic instability, as these cells appear to make many mistakes that would be expected to activate the NoCut pathway. Genomic instability is an emerging driver and target in human cancer therefore the proposed work is positioned at the cutting-edge of research, with clear long-term medical and biotechnological potential. Since cancer effects around 2.9 million people in Europe each year, and the global spending on cancer therapy is around $100 billion (IMS Institute for Healthcare Informatics states), progress in this area is likely to have a direct impact on both human health and the European economy.
Working model of the mechanism used by the NoCut checkpoint