Abstract
Archaea employ Orc1-2, an Orc1/Cdc6 family protein, to mediate DNA damage-responsive (DDR) regulation, which orchestrates a series of cellular responses to DNA damage. However, how the DDR process is regulated remains elusive. To investigate whether the Orc1-2 functions could be regulated by posttranslational modifications (PTMs), differential PTMs were analyzed for Orc1-2 proteins in cells of normal growth versus those in DNA damage-treated cells. We found only Orc1-2 proteins present in untreated cells are phosphorylated at T356. Since T356 is located in the DNA-binding pocket of the archaeal DDR regulator in the predicted structure, its phosphorylation may impair the DNA binding of the protein. Indeed, characterization of T356A, the phospho-ablative form, and T356D, the phospho-mimetic form of Orc1-2, revealed that only the phospho-ablative form retained the specific DNA binding. Genetic characterization and RNA-seq analyses further revealed that their corresponding mutants also exhibited expected phenotypes: orc1-2T356D no longer exhibited DNA damage responses upon NQO treatment, while the phospho-ablative mutant orc1-2T356A is not only more tolerant to DNA damage agents but also prolongs the window of the DNA damage response. Taken together, these results indicated that T356 phosphorylation deactivates Orc1-2, thereby attenuating the archaeal DNA damage response.