Merrick, Catherine Jill; (2004) Visualisation of altered replication dynamics during the S phase checkpoint response to DNA damage in human cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Eukaryotic cells respond to DNA damage within S phase by activating an intra-S phase checkpoint; a response which includes reducing the rate of DNA synthesis. In yeast cells this occurs via a checkpoint-dependent inhibition of origin firing and stabilisation of ongoing forks, together with a checkpoint- independent slowing of fork movement. In higher eukaryotes, however, the mechanism by which DNA synthesis is reduced is less clear. This work describes DNA fibre labelling strategies that offer a quantitative assessment of rates of replication fork movement, origin firing and fork stalling throughout the genome by examining large numbers of individually labelled replication forks. It shows that exposing S phase cells to ionising radiation (IR) induces a transient block to origin firing but does not affect fork rate or fork stalling. Exposure to alkylating agents or UV light causes a slowing of fork movement and a high rate of fork stalling in addition to a sustained block to origin firing. Nucleotide depletion also reduces fork rate, increases stalling and suppresses new origin firing. The block to new origin firing depends on the central checkpoint kinases ATM and ATR in response to damage by IR and UV respectively. Both responses are transduced jointly by the CHK1 and CHK2 kinases. ATR also has a role in preventing irreversible fork stalling but this appears to be independent of CHK1. Finally, the slowing of replication forks is independent of both ATR and CHK1. Thus, this work provides a detailed picture of the mechanics of the replication response to DNA damage in human cells, and clarifies the relative checkpoint dependencies of each aspect of this response.

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