Elucidation of checkpoint kinase interactions with the replisome
Lead Research Organisation:
University of Cambridge
Department Name: Biochemistry
Abstract
Cells must faithfully and accurately duplicate their genome during S-phase. The DNA replication checkpoint (DRC) is a kinase signalling pathway that ensures the coordination of DNA replication, cell cycle progression and DNA damage repair during replication stress. This checkpoint pathway is a major area of investigation for the discovery of new chemotherapeutics that can selectively kill cancer cells.
One of the major functions of the DRC checkpoint kinases (ATR-Chk1 in humans) is to prevent replication fork collapse. However, despite their involvement in fork integrity, it is largely unknown how these kinases regulate the replisome and how this prevents irreversible fork collapse during replicative stress. This project will address the following:
1. How do checkpoint kinases interact with the replisome?
2. How do stalled and normal forks differ?
3. How do these interactions regulate replication fork integrity?
I have taken advantage of the unparalleled genetic and molecular biology tools available in budding yeast to identify novel checkpoint-replisome interactions, which I will use as a platform to understand checkpoint function in human cells. To identify replisome-checkpoint interactions in an unbiased way, I have used in vivo biotinylation and mass-spec strategies to compare how these interactions vary with different conditions, such as during normal replication versus during activation of the DRC. From these studies I have identified novel mechanisms through which the checkpoint controls replication fork integrity. I aim now to translate these studies from yeast to human cells to determine whether these discoveries may affect our understanding of how checkpoint inhibitors kill cancer cells.
One of the major functions of the DRC checkpoint kinases (ATR-Chk1 in humans) is to prevent replication fork collapse. However, despite their involvement in fork integrity, it is largely unknown how these kinases regulate the replisome and how this prevents irreversible fork collapse during replicative stress. This project will address the following:
1. How do checkpoint kinases interact with the replisome?
2. How do stalled and normal forks differ?
3. How do these interactions regulate replication fork integrity?
I have taken advantage of the unparalleled genetic and molecular biology tools available in budding yeast to identify novel checkpoint-replisome interactions, which I will use as a platform to understand checkpoint function in human cells. To identify replisome-checkpoint interactions in an unbiased way, I have used in vivo biotinylation and mass-spec strategies to compare how these interactions vary with different conditions, such as during normal replication versus during activation of the DRC. From these studies I have identified novel mechanisms through which the checkpoint controls replication fork integrity. I aim now to translate these studies from yeast to human cells to determine whether these discoveries may affect our understanding of how checkpoint inhibitors kill cancer cells.