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DNA DAMAGE RESPONSES IN YEAST AND HUMAN CELLS
Stalled replication forks activate the Mec1/Rad53 checkpoint pathway (green arrows). This pathway inhibits the activation of late-firing replication origins (red lines) and stabilises stalled replication forks (green arrows).
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Regulation of DNA replication by the DNA damage checkpoint
We are interested in how DNA damage checkpoint pathways regulate DNA replication. We have found that when DNA damage or DNA replication fork stalling activates the checkpoint kinases Mec1 and Rad53, at least two aspects of DNA replication are regulated. Firstly, the activation of late-firing replication origins is inhibited (12, 14). Secondly, DNA replication forks are stabilised (29). This role for the checkpoint pathway in stabilising DNA replication forks appears to be especially critical for maintaining viability (33).
ONGOING PROJECTS:
Checkpoint regulation of DNA replication in human cells
To complement our work in yeast we have begun to analyse the role of the DNA damage checkpoint pathways in regulating DNA replication in human cells. We have established an assay to quantify a number of replication parameters including rates of origin firing, fork progression and fork stalling. Our preliminary analysis of replication parameters after a variety of DNA damage can be found in ref. 38 (Catherine Merrick). We are currently extending this analysis to include cells with compromised checkpoints using siRNA as well as a number of knockout cell lines. Catherine will soon be finishing her PhD. Anyone interested in continuing this line of research with us should contact John Diffley.
An example of DNA fibre analysis of DNA replication in human cells. Cells were labelled sequentially with iododeoxyuridine and chlorodeoxyuridine, DNA was spread on microscope slides and stained with specific antibodies for the halogenated nucleosides (IdU, red; CldU, green). This figure shows examples of ongoing replication forks (1), new initiations (2a and b) and termination (3). See Merrick et al. 2004 for details.
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Checkpoint regulation of replication forks in yeast
Our previous work indicated that the extreme sensitivity of mec1 and rad53 mutants to DNA damaging agents like methyl methanesulfonate (MMS) was due primarily to their inability to stabilise stalled replication forks. We are currently trying to understand how forks are stabilised by the checkpoint pathway by looking for new Rad53 substrates. To this end, we have epitope tagged a large number of proteins involved in DNA replication and DNA repair and have screened for proteins that are phosphorylated at early time points after DNA damage (Philip Zegerman). We have identified some candidate substrates and are currently characterising their role in replication fork stabilisation. We are also using a genetic approach by screening for mutants that increase the viability of rad53 mutants after treatment with hydroxyurea and MMS (Monica Segurado Carrascal).
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