Effect of phosphorylation of the intrinsically disordered domain of A. thaliana SOG1 on its conformation and DNA binding

DNA damage leads to abnormalities and diseases. Therefore, every organism needs to protect its DNA integrity. Plants and animals achieve this through the DNA Damage Response (DDR) pathway. In animals, this pathway is centrally controlled by the well-known p53 protein. In plants, the intrinsically disordered transcription factor SOG1 functions as the master regulator of the DDR in which it is responsible for regulating DNA repair, cell cycle arrest and apoptosis upon genotoxic stress.
SOG1 belongs to the plant-specific NAC transcription factor family and consist of a small N-terminal domain (NTD), a NAC domain and a large C-terminal domain (CTD). The NAC domain is a conserved domain responsible for DNA binding and dimerization. The CTD is intrinsically disordered and functions as a transcriptional regulatory region (TRR). SOG1’s transcriptional activity is activated in planta by phosphorylation of the TRR by different kinases depending on the type of stress.
For this poster, the effect of reported in planta phosphorylation states of the CTD on the overall conformation and DNA binding of A. thaliana SOG1 is investigated. For this, two phospho-mimic mutants are used as well as the non-phosphorylated, WT protein.
CD and analytical SEC were performed to detect changes in secondary structure composition and hydrodynamic radius respectively. These will show if phosphorylation of the CTD can induce structural changes which can function as an interaction site for target proteins/DNA. Both experiments are supplemented by SAXS of the individual proteins to determine their overall shape and to confirm the acquired data.
ITC was performed using a brca1 promotor and a randomly generated DNA fragment. This experiment will give insights into the specificity and the affinity of each protein and into a possible role of the in planta phosphorylation state on DNA binding.