Rationale design of unrestricted pRN1 derivatives and their application in construction of a dual plasmid vector system for Saccharolobus islandicus

Sulfolobus species belonging to crenarchaea code for a novel DNA damage response (DDR) system in which an Orc1/Cdc6 family protein, Orc1-2, initiates a global cellular response to DNA damage by functioning as a global transcriptional regulator. However, the mechanism that modulates Orc1-2 activity upon DNA damage remains unclear. In this study, we found that Sulfolobus islandicus Orc1-2 was phosphorylated under normal growth and non-phosphorylated upon DNA damage treatment at Thr356. Interestingly, the phospho-mimetic mutant orc1-2T356D exhibit defective DDR processes, while the non-phosphorylatable mutant orc1-2T356A shows prolonged activation of DDR in which the expression of representatives DDR genes are significantly elevated. DNA binding assays revealed that the Orc1-2T356D protein showed reduced DNA binding activity. In contrast, the Orc1-2T356A protein shows enhanced chromosome binding. These data indicate that Orc1-2 phosphorylation negatively regulates the DNA damage response in Sulfolobus by modulating the DNA binding activity of the core regulator.