DNA damage response (DDR) is essential for the maintenance of genome integrity in all three domains of life, and the process is controlled by evolutionarily unrelated factors in Bacteria and in Eukarya. While DDR is primarily mediated by cleavage of the global repressor, LexA in the former, the process in the latter is mainly orchestrated by two evolutionally conserved kinases, ATM/ATR. Strikingly, none of these DDR regulators have a homologue in Archaea. As a result, it remains elusive as to how organisms in Archaea coordinate cellular processes in response to DNA damage signal (s). Nevertheless, investigation of genome expression upon UV light exposure in Sulfolobus species revealed a number of differentially expressed genes including genes encoding a paralogue of TFB protein (TFB3) and an orthologue of Orc1/Cdc6 protein (Orc1-2). Here, we apply a combination of genetic, biochemical, transcriptome and phylogenetic analysis to investigate their possible roles in archaeal DDR using Sulfolobus islandicus REY15A as the model.
Firstly, we constructed tfb3 gene deletion mutant and the transcriptome analysis of the resulting mutant (Δtfb3) revealed that TFB3 is essential for the transcriptional activation of a subset of DDR genes. Phenotypic characterization of Δtfb3 showed that the mutant loses its ability to form cell aggregates upon DNA damage and is moderately sensitive to DNA damage. Interestingly, CHIP-qPCR analysis showed that TFB3 specifically binds to the promoter region of TFB3-dependent genes, suggesting that TFB3 directly modulates the transcriptional process upon DNA damage. Further, mutagenesis of the TFB3 protein and subsequent functional analysis indicated that the N terminal Zn ribbon and C terminal Coiled-Coil motif are essential for its function in the transcriptional activation. Furthermore, the phylogenetic analysis revealed a co-evolution of TFB3 with its target system (Ced, the Crenarchaeal system for exchange of DNA), suggesting that the TFB3- mediated transcriptional regulation may represent a well conserved DDR regulatory circuit for intercellular DNA transfer in Crenarchaeota.
Then, we showed that the previously constructed orc1-2 deletion mutant (Δorc1-2) is hypersensitive to NQO treatment and the transcriptome analysis of the mutant revealed that Orc1-2 is essential for the global transcriptional regulation upon DNA damage. Orc1-2- dependent processes include the TFB3-controlled DNA transfer pathway, DNA replication initiation, cell cycle arrest and potential translesion DNA synthesis. Consistently, Δorc1-2 is defective in DNA damage induced cell aggregation and cell cycle control. Furthermore, DNase I footprinting assay with Orc1-2 indicated that this protein is capable of protecting a conserved promoter element present in a number of Orc1-2-dependent genes and reporter gene assay demonstrated that this motif is responsible for the DNA-damage responsive expression, suggesting that Orc1-2 binds to the conserved DNA damage responsive element (DDRE) upon DNA damage and modulates transcriptions of DDR genes. Eventually, a promoter switch strain was constructed and analysis of the DDR in this strain showed that the induction of Orc1-2 is essential but not sufficient for activation of DDR, suggesting Orc1-2 could be posttranslationally modified upon DNA damage.