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Deciphering the mechanism of irreversible replication fork arrest

Project description

The mechanism of irreversible replication fork arrest

DNA replication is essential for cell proliferation, and obstacles to this process result in replication stress blocking replication forks. In response, cells activate the DNA damage checkpoint pathway to prevent DNA damage and ensure cell survival. The essential function of the checkpoint is to stabilise blocked replication forks to ensure that replication will resume after obstacles are removed. The yeast checkpoint kinase Rad53 and its human analogue Chk1 are involved in the prevention of irreversible replication fork arrest (IRFA) and cell death caused by replication stress. The EU-funded Irrev Fork Arrest project will employ in vitro replication reconstitution with yeast purified proteins to determine the proteins and enzymatic activities required to promote IRFA in the absence of the checkpoint.

Objective

DNA replication is essential for cell proliferation. Obstacles to replication generate replication stress by stalling replication forks. In response to replication stress, cells activate the DNA damage checkpoint pathway that coordinates a cellular response to prevent DNA damage and ensure cell survival. One essential function of the checkpoint is to stabilise stalled replication forks and ensure that replication will resume after obstacles are removed. Specifically, the yeast checkpoint effector kinase Rad53 and its human counterpart Chk1 are essential to prevent irreversible replication fork arrest (IRFA), DNA damage and cell death under replication stress. A screen to identify factors required to promote IRFA has revealed a role for unrestricted recombination events in promoting IRFA. However, mechanistic studies are still required to understand how the checkpoint stabilises stalled forks and prevents IRFA. Recently, in vitro replication reconstitution with yeast purified proteins in the lab has helped uncover important mechanisms of DNA replication. I will use this system to reconstitute IRFA in vitro and determine the proteins and enzymatic activities required to promote IRFA in the absence of the checkpoint. I will then look for phosphorylation targets of Rad53 to understand how the checkpoint prevents IRFA. I will study changes at the DNA and replisome of stalled replication forks by 2D electrophoresis, mass spectrometry and cryo-EM to understand the causes of the irreversibility of IRFA. We seek to better characterise an essential function of the DNA damage checkpoint and define a new role for unrestricted recombination in promoting DNA damage. Main challenges of current cancer therapies include the appearance of surviving checkpoint-deficient cancer cells. Despite constant replication stress, it is unknown why these cells do not suffer irreversible fork arrest. Understanding IRFA could help design new therapies to target checkpoint-deficient cancer cells.

Coordinator

THE FRANCIS CRICK INSTITUTE LIMITED
Net EU contribution
€ 212 933,76
Address
1 MIDLAND ROAD
NW1 1AT London
United Kingdom

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Region
London Inner London — West Camden and City of London
Activity type
Research Organisations
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Total cost
€ 212 933,76