Analysis of the DNA damage response in model systems
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The integrity of our DNA is constantly threatened by endogenous and exogenous sources of damage. In the absence of sufficient repair mechanisms, this damage can lead to mutations, potentially resulting in the development of cancer. For this reason, our cells have evolved a mechanism, termed the DNA damage response to correct DNA damage and prevent its propagation to further cell generations. ATM is central to the DDR. In our lab, a proteomic study of ATM was carried out to identify new ATM binding partners. Among the candidates identified was ZC3H11A, a member of the C3H1 class of zinc finger proteins previously identified as a potential ATM substrate after IR. Here we show that ZC3H11A is required for ATM autophosphorylation following DNA damage. To examine the effect of ZC3H11A depletion on ATM function, we examined checkpoint activation and DNA repair. While ZC3H11A was dispensible for G2/M checkpoint activation and NHEJ, it was essential for BRCA1 recruitment and Homologous recombination. We also show that ZC3H11A resides in the heterochromatic compartment of DNA and is required for the relaxation of heterochromatin in response to IR. Using bioinformatic analysis, we identified several new potential PIK kinase phosphorylation sites on ZC3H11A. Two of these sites are highly conserved, suggesting that they are important for ZC3H11A function. We also identified a highly structured, heavily phosphorylated C-terminal region and a coiled coil region within the ATM interacting domain. These findings provide the bases for future structure-function studies of the ZC3H11A protein. The DDR is highly conserved from yeast to human. Rad9 is the budding yeast DDR mediator and is required for checkpoint activation and DNA repair. Rad9 is heavily phopshorylated and much of this phosphorylation is believed to be cell cycle regulated. To understand Rad9 phosphorylation in the context of cell cycle timing, we performed mass spectrometric analysis on purified Rad9 from G1 and G2-arrested cells. Here we present 22 previously unidentified phosphorylation events on Rad9. Within the Chk1 activating domain of Rad9 we identified two sites, T125 and T143 which differentially regulate the Rad9-Chk1 interaction.