| Summary: | The ATM and ATR protein kinases play central roles in the cellular response to DNA damage. The full scope of the DNA damage response (DDR) is not known and there is increasing evidence that ATM and ATR signalling targets many cellular processes. Identifying novel substrates of the DDR is important to improve understanding of the cellular events that occur following DNA damage and may be of therapeutic use; components of the DDR can be targeted by drugs to sensitise cancer cells to radiation or chemotherapy. Proteomic screens to identify novel targets of the DDR have been enriched for RNA processing factors, and post-transcriptional control of gene expression is emerging as an important contributor to the preservation of genome stability. In this thesis we describe the identification of the Xenopus laevis Cip29 protein as a novel target of the DDR. Cip29 is a highly conserved protein and in human cells has been shown to interact with several proteins required for mRNA processing and nuclear export. We found that XCip29 is phosphorylated in Xenopus egg extract in the presence of DNA templates that trigger a DNA damage checkpoint response. Moreover we established that ATM phosphorylates XCip29, and mapped the phosphorylation to amino acid ser95. In the course of our analyses we also identified a consensus motif for cyclin-dependent kinases within the XCip29 amino acid sequence at serl62. We found thatXCip29 is phosphorylated in mitotic Xenopus egg extract at this consensus site, and that phosphorylation of XCip29 at serl62 involves the activity of one of the major mitotic kinases, Cdkl. We also investigated XCip29 protein-protein interactions in Xenopus extract as we thought this may provide insight into XCip29 protein function. Mass spectrometry analyses revealed multiple putative XCip29-interacting proteins; however of the proteins we selected to investigate further we were unable to detect interactions with XCip29 by co-immunoprecipitation.
|
|---|
