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Impact of chromatin context on DNA double-strand break repair kinetics, fidelity and signaling

Projectomschrijving

Kanker ontstaat door permanente schade aan het DNA. Schade aan het DNA komt veelvuldig voor, maar cellen kunnen doorgaans de schade snel opsporen en herstellen. Echter, herstel van schade is niet altijd optimaal en het wordt steeds duidelijker dat de verpakking van DNA in chromatine bepaalt hoe cellen omgaan met schade, zoals breuken in het DNA. In dit project willen we met een reeks nieuw ontwikkelde technieken gaan onderzoeken hoe het chromatine de efficiëntie van schadeherstel beïnvloed, en welke gevolgen dit heeft voor de genetische stabiliteit van een cel. Met deze nieuwe technieken kunnen we de kinetiek van DNA herstel op een willekeurige plek in het genoom nauwkeurig bepalen, en bestuderen in relatie tot het effect van de schade-response op het verdere gedrag van de cel. Met de verworven inzichten wordt het mogelijk om de uitkomst van de schade-response voor het (dis)functioneren van een cel beter te voorspellen.

Producten

Titel: Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance
Auteur: Ruben Schep, Eva K. Brinkman, Christ Leemans, Xabier Vergara, Ben Morris, Tom van Schaik, Stefano G. Manzo, Daniel Peric-Hupkes, Jeroen van den Berg, Roderick L. Beijersbergen, René H. Medema, Bas van Steensel
Link: https://doi.org/10.1101/2020.05.05.078436
Titel: Cell fate decisions after DNA damage; the role of dose versus location of the damage
Auteur: Jeroen van den Berg Rene H. Medema
Titel: Chromatin Context in the DNA Damage Response
Auteur: Jeroen van den Berg Rene H. Medema
Titel: Seminar Cambridge Epigenetics Club, Cambridge UK.
Auteur: Bas van Steensel
Titel: A limited number of double-strand DNA breaks is sufficient to delay cell cycle progression
Auteur: van den Berg, Jeroen, G. Manjón, Anna, Kielbassa, Karoline, Feringa, Femke M, Freire, Raimundo, Medema, René H
Magazine: Nucleic Acids Research

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Samenvatting van de aanvraag

DNA double-strand breaks (DSBs) occur very frequently in human cells, and it is essential that such breaks are quickly detected and repaired. Progression into mitosis with unrepaired breaks can compromise genomic integrity and viability, and these deleterious effects of DNA damage are frequently exploited in anti-cancer therapies, such as radiotherapy and a large variety of chemotherapeutic agents. It is becoming increasingly clear that the packaging of DNA into chromatin affects how cells cope with DSBs. Because chromatin structure and composition varies widely across the genome, signalling and repair of DSBs in different loci may have very different efficiencies. Indeed, we have evidence that the error rate of NHEJ is dependent on the genomic location and that location of the damage can affect outcome of a damaging insult, but the underlying principles are not known and we currently lack the knowledge that allows us to predict how a cell will deal with a DNA lesion at any specific site. In this project, we aim to resolve the influence of chromatin context on DNA repair and the outcome of the DNA damage response. For this, we have developed novel tools that enable us to create DSBs in a tightly controlled manner at endogenous genomic loci of choice. In parallel, we have developed new assays that allow us to rapidly and precisely measure the kinetics of repair at DNA sequence level, and to monitor the response of single cells using a set of live-cell reporters. Using these tools, we aim to understand exactly how the chromatin context of a DSB determines the kinetics and fidelity of repair, and how this is linked to subsequent cell fate decisions. Our key objectives are: 1. To obtain a quantitative understanding of the effects of chromatin context on NHEJ repair rates and fidelity. For this, we will induce breaks at defined loci with different chromatin environments, and precisely measure the kinetics of NHEJ-mediated repair and fidelity, using newly developed molecular assays that directly quantify broken and repaired DNA. By quantitative computational modelling we can accurately determine kinetic parameters and unravel how local chromatin environment affects repair. 2. To understand how chromatin context affects DSB signalling. For this, we will use a set of specially designed fluorescent reporters to determine the kinetics of the DNA damage signalling and the local assembly of repair proteins on DSBs in living cells. 3. Reveal how chromatin context of DSBs determines cell fate decisions. For this, we will determine how breaks at hundreds of different genomic locations can lead to a distinct DNA damage response in terms of checkpoint activation, repair and outcome. 4. Reveal how chromatin context can be modified to alter the DNA damage response. Using the insights obtained in Objectives 1-3 we will select candidate chromatin modifications that we want to (locally) alter to study if this affects DSB repair kinetics, fidelity, signalling and cell fate decisions. The results obtained in this project are expected to yield for the first time quantitative estimates of the repair rates and fidelities, damage signalling and cell fate decisions triggered by DSBs in dozens of genomic loci in human cells, and will provide fundamental insights into the effects of local chromatin context on these processes. This work can help us better understand and predict the outcome of a DNA damaging insult, and may lay the basis for more selective interventions in tumor cell proliferation.

Onderwerpen

Kenmerken

Projectnummer:
91215067
Looptijd: 100%
Looptijd: 100 %
2016
2021
Onderdeel van programma:
Gerelateerde subsidieronde:
Projectleider en penvoerder:
Prof. dr. R.H. Medema
Verantwoordelijke organisatie:
Nederlands Kanker Instituut