Molecular basis of DNA recognition by Arabidopsis thaliana SOG1: The master regulator for DNA damage control in plants

SOG1 is the master regulator of the DNA damage response (DDR) in plants, a large signal transducing pathway responsible for sensing DNA damage and maintaining genomic integrity. Upon DNA damage, SOG1 regulates the expression of over 300 genes involved in DNA repair, cell cycle arrest, apoptosis and endoreduplication. SOG1 belongs to the NAC family, a large plant-specific family of transcription factors involved in stress response and development. NAC proteins are characterized by a conserved N-terminal NAC domain, which mediates DNA binding and dimerization, and a large, intrinsically disordered C-terminal domain (CTD), which functions as the transcriptional regulatory region (TRR). In contrast to most NAC proteins, SOG1 also contains a small N-terminal domain (NTD) with unknown function. Despite the crucial role of SOG1 in protecting genome integrity, the protein is still poorly understood, especially information on its structure-function relationship is lacking. The main goal of this thesis is to understand the molecular basis of the DNA specificity of A. thaliana SOG1 in order to gain more insight into how SOG1 recognizes its target genes and regulates transcription. The full length A. thaliana SOG1 protein was obtained from expression in E. coli using a SUMO solubility tag, which prevented aggregation during expression without tag. EMSA and ITC experiments with the isolated SOG1 NAC domain confirmed that this domain is responsible for dimerization and DNA binding, serving as the primary interface that determines sequence specificity. The NTD domain of SOG1 likely does not function in DNA binding, since it does not impact DNA specificity and only modestly reduces
binding affinity. The transcriptional activity of SOG1 is regulated in vivo through phosphorylation of its CTD. EMSA and ITC using WT, non-phosphorylated SOG1 and phospho-mimicking mutants revealed that the negatively charged CTD indirectly enhances DNA selectivity, but further does not contribute to DNA binding. Neither is its phosphorylation state involved in modulating DNA interaction. SAXS further showed that the CTD remains highly flexible in solution even when SOG1 binds DNA,
maintaining accessibility of its phosphorylation region. Finally, the structure of the SOG1 NAC domain in complex with target DNA was resolved by single-particle cryo-electron microscopy (EM) at a resolution of 2.97 Å, while bound to a megabody against the NAC domain. The structure of SOG1 NAC domain consists of a twisted β-sheet flanked by α-helixes, which is characteristic for the NAC family. The Lys153, Thr154 and Gly155 residues within the conserved WHKTGRT motif were found to mediate
sequence specific interactions with the established DNA target site, contributing to DNA binding specificity. These residues are located on the β-strand that intercalates with the DNA major groove, a binding mechanism observed for other NAC members. In conclusion, A. thaliana SOG1 functions as a typical NAC transcription factor by directly binding DNA through its dimerized NAC domain, which mediates sequence specificity. Its transcriptional activity is regulated by phosphorylation of the CTD,
which does not alter DNA binding, but likely influences interactions with regulatory partners or its liquid-liquid phase separation (LLPS) behaviour to modulate target gene expression.