Archaea constitutes the third domain of life. Comparative genomic analyses have revealed that the archaeal DNA replication machinery shares similarity with that of eukaryotes and can generally be considered a simplified version of the eukaryotic machinery. For example, archaeal Orc1 paralogues show a high sequence similarity to the largest subunit of origin recognition complex (Orc1) and to the cell division cycle protein 6 (Cdc6). Sulfolobus species contain three orc1 genes, orc1-1, orc1-2 and orc1-3. In S. islandicus, orc1-1 and orc1-3 code for replication initiators, whereas deletion of orc1-2 gene does not influence the origin firing in this archaeon. Nevertheless, investigations of ultraviolet (UV) irradiation response in S. solfataricus by transcriptome analysis indicated that UV treatment imposes differential gene expression on the two categories of orc1 genes: while the expression of the initiator orc1 genes are down regulated, the expression of the non-initiator orc1-2 is up regulated. Here, we applied a combination of genetic, transcriptome and biochemical analyses to investigate the functions of Orc1-2 and its conserved domains in DNA damage response (DDR) in S. islandicus.
At first, the studies of the effects of 4-nitroquinoline 1-oxide (NQO) on wild type (WT) strain E233S and orc1-2 deletion mutant (Δorc1-2) showed that Δorc1-2 is hypersensitive to NQO treatment and loses the capability of DNA damage-induced cellular aggregation and cell cycle control. The transcriptome analysis revealed that Orc1-2 is essential for the NQOresponsive expression. Reporter gene and DNase I footprinting assays confirmed that Orc1- 2 binds a previously reported conserved DNA damage responsive element (DDRE) present in the promoters of many up-regulated DDR genes and regulates their expression. Moreover, manipulation of orc1-2 expression by promoter switch experiments demonstrated that a high level of orc1-2 expression is essential but not sufficient to trigger DDR.
Next, a series of Orc1-2 mutants were conducted, including truncation derivatives and substitution mutants of highly conserved amino acids. Electrophoretic mobility shift assays (EMSA) were used to test the affinity and specificity of Orc1-2 mutants for the DDRE motif in the promoter of DDR genes in vitro. Orc1-2 mutants constructed based on the CRISPRassisted gene editing method were used to study the functions of the Orc1-2 conserved amino acids and domains in vivo. We found that: (a) both N-terminal AAA+ ATPase domain and C-terminal winged-helix (WH) domain are essential for Orc1-2-mediated DDR regulation. (b) The ATP-binding deficient Walker A (WA) mutant considerably decreased the level of DNA binding in vitro and was proved to be essential for the function of Orc1-2 in regulating DDR in vivo. (c) The initiator specific motif (ISM) is also essential and EMSA results indicated that it may be involved in the recognition of DDRE and stabilizing the binding complex. (c) Expression of a Walker B mutated Orc1-2 which could bind but fail to hydrolyze ATP causes cell death and formation of enlarged cells in S. islandicus. (d) Four conserved arginine residues in the WH domain function in DNA binding and two of them (R381 and R383) interact with the DDRE motif. (e) A CTCTCT motif was identified in the promoter of orc1-2 and dpo2 and could influence their gene expression under physiological growth condition. Together, an Orc1-2-centered network of DNA damage response in S. islandicus was revealed.