DNA replication forks and checkpoints

The DNA replication checkpoint senses perturbation of DNA replication forks by a wide variety of agents.

We know the protein components and the overall architecture of the checkpoint pathways, but we do not understand how very different fork stalling events can signal via a common pathway.

The first aim of this proposal is to use the fully reconstituted DNA replication system we have developed together with purified checkpoint proteins and defined DNA templates to elucidate in molecular detail the mechanisms by which the checkpoint pathway is activated. One of the main functions of this checkpoint is to prevent irreversible replication fork arrest (IRFA).

From a whole genome siRNA screen, we have recently found that loss of the HLTF DNA translocase suppresses IRFA.

Furthermore, we have found evidence that HLTF generates a toxic recombination intermediate and that this is conserved in yeast.

Our second aim is to use genetics in human cells along with biochemistry with yeast proteins to understand how IRFA is generated and how the DNA damage checkpoint protects forks from IRFA.