A number of agents can damage DNA, resulting in mutations that may contribute to cancer. Therefore, the cell has evolved response pathways to repair damaged DNA. The DNA damage response involves recognition of DNA lesions and, subsequently, activation of downstream effectors via phosphorylation cascades. The downstream effectors include transcription factors that regulate the expression of DNA damage response proteins. A recent study identified 30 transcription factors that may regulate gene expression in response to the DNA damaging agent, methyl methanesulfonate (MMS), in yeast. Two of these proteins, Swi4 and Swi6, regulate expression of genes involved in cell cycle progression. Determining how Swi4 and Swi6 are regulated by phosphorylation during MMS treatment and how they, in turn, regulate target gene expression in response to MMS treatment will provide us with significant insight into the role of transcription in the cellular DNA damage response. Given the hypothesis that phosphorylation regulates the transcriptional activity of Swi4 and Swi6 in response to DNA damage, this project aims to determine the role of phosphorylation of Swi4 and Swi6 in the DNA damage response by addressing the following specific aims: (1) Identify the Swi4 and Swi6 phosphorylation sites that are differentially regulated during MMS treatment. (2) Determine the functional relevance of phosphorylation of Swi 4 and Swi6 to the cellular DNA damage response. (3) Determine the protein kinases and phosphatases responsible for phosphorylation of Swi4 and Swi6 during MMS treatment. The MMS-specific phosphorylation sites regulating each transcription factor will by determined by using mass spectroscopy to identify phosphopeptides present in yeast cultures treated with MMS but absent in untreated cultures and vice versa. To determine the roles of these phosphorylation sites in the response to MMS-induced DNA damage, phosphorylation site mutants will be tested for defects in growth, survival, and DNA damage repair upon MMS treatment, and the effect of mutating these sites on the expression of downstream transcriptional targets will be determined. To determine which protein kinases and phosphatases regulate phosphorylation of these sites, the phosphorylation state of each site in will be evaluated in candidate kinase/phosphatase mutant yeast.
Since DNA damage response pathways are conserved between yeast and humans, this study will provide us with information regarding the ways cells respond to and correct the mutations that can lead to cancer and, thus, regarding the cellular processes that may prevent and/or may be disrupted in cancer ? ?
| Jaehnig, Eric J; Kuo, Dwight; Hombauer, Hans et al. (2013) Checkpoint kinases regulate a global network of transcription factors in response to DNA damage. Cell Rep 4:174-88 |
