Cellular responses to DNA damage, studies on the functional organization of the S phase checkpoint proteins at sites of stalled DNA replication
Supervisor: Dr Clive Price
The maintenance of genome stability is critical to the success of cell division. Loss of control of genome stability results in the accumulation of DNA damage, acquisition of mutation and structural re-arrangement. The DNA structure integrity checkpoint control pathways act to block cell cycle progression in response to DNA damage and the inhibition of DNA replication and serve as a primary mechanism for the maintenance of genome stability. Activation of the checkpoint pathways has been implicated in the early stages of tumouriogenesis and it has been persuasively argued that defects in DNA checkpoints pathways contribute significantly to the development of all cancers. The project is designed to describe the order and dependency of assembly of DNA replication checkpoint factors at stalled replication forks, the core technique being chromatin immunoprecipitation (ChIP). Using fission yeast as a model system it is proposed to identify the requirements for loading of the ATR/ATRIP, the 9-1-1, Rad17-Rfc complexes at sites of stalled replication forks with a focus on the role of Rad4TopBP1 in this process. It rests on the preliminary data indicating that Rad17p is not required for Rad4TopBP1 loading at stalled replication forks, whilst Rad9p function is required. This observation calls into question the paradigmatic view of the organization of these factors in response to genotoxic challenge, contrasting with previous observations indicating that 9-1-1 loading is Rad17p dependent. In parallel protein-protein interactions between the checkpoint proteins will be tested by co-immunoprecipitation experiments in the context of replication inhibition and the structure of the stalled replication fork examined by 2 dimensional agarose gel electrophoresis. The experimental programme will provide a comprehensive description of the behaviour of the checkpoint proteins at stalled replication forks that will provide new mechanistic insights.