Phosphorylation And Multi-level Regulation Of Orc1-2,the Archaeal Global DNA Damage-responsive Regulator

Organisms of all three domains of life constantly face damages on their genetic materials.They have evolved a sophisticated signal transduction network,known as DNA damage response（DDR）to safeguard genomic integrity by coordinating a cascade of cellular events,including DNA repair,damage tolerance,inhibition of DNA replication,and cell cycle arrest.The defective DDR process will cause severe diseases such as immune deficiency,neurological degeneration,premature aging and cancer of human beings and other higher eukaryotes,and lead to gene mutation or cell death of prokaryotes.Therefore,strict spatiotemporal regulation of DDR is required.Extensive studies of DDR in bacterial,eukaryotic,and archaeal model organisms have revealed that they utilize distinctive regulatory mechanisms.Bacteria primarily employ the proteolysis of LexA,a global transcriptional repressor of bacterial SOS response,to liberate the expression of a large number of genes involved in cell cycle arrest and DNA repair.Eukaryotes have adopted a cascade of phosphorylation on key factors that are mediated by three related kinases（DNA-PK,ATM,and ATR）to trigger a global response upon different types of DNA damage.For Archaea,however,it has been demonstrated that Orc1-2,an ortholog of the archaeal/eukaryotic origin recognition complex（Orc）proteins,plays a central role in DDR regulation.However,the involved mechanism for Orc1-2 regulation in Archaea remains to be studied.This paper focuses on Orc1-2,the archaeal global DNA damage-responsive regulator,to explore the mystery of DDR regulation of Archaea from the perspectives of protein phosphorylation and functional domains.Reversible protein phosphorylation is the main mechanism of DNA damage signal transduction in eukaryotes.It has also been found in Archaea to participate in the regulation of cell motility,transcription,translation and other cellular activities.Studies on biochemical characteristics and phosphoproteome showed that archaeal Orc1 proteins could be phosphorylated.Therefore,we propose the hypothesis that Orc1-2 is likely to achieve functional regulation through post-translational modifications（PTMs）such as phosphorylation.To determine whether Orc1-2 undergoes PTMs in the process of DDR,and to explain the effect of PTMs on the Orc1-2 functional regulation,we constructed a strain in which a 6x His-tag coding sequence was inserted into the 3’ end of the genomic orcl-2 gene and evaluated the phenotypes of the strain.We found that His-tagged Orc1-2 in this strain should reflect the function of the native Orc1-2.Next,the tagged Orc1-2 proteins from drugtreated and untreated samples were isolated by His-tag affinity chromatography individually.LC-MS/MS analysis of purified proteins identified a phosphorylated site at Thr356 of Orc1-2 in untreated cells,but not in NQO-treated cells.To investigate how does phosphorylation affect the function of Orc1-2 during DDR,we constructed non-phosphorylatable orc1-2T356A and phospho-mimetic orc1-2T356D mutants individually by introducing site mutagenesis at the genomic orcl-2 locus.Genetic analysis of the two mutants showed that the phospho-mimetic mutant S.islandicus orcl-2T356D is hypersensitive to DNA damage.Importantly,orc1-2T356D no longer exhibit DDR upon NQO treatment.However,the non-phosphorylatable mutant orcl-2T356A has increased tolerance to DNA damage,higher levels of DNA damage induced expression of representative DDR genes,and longer DDR windows.These results suggest that,the phosphorylation of Orc1-2 at Thr356 negatively regulates the DDR in S.islandicus.To provide mechanistic insight into how Orcl-2 phosphorylation affected its function,we purified two mutant proteins Orcl-2T356A and Orcl-2T356D from E.coli and then performed in vitro DNA binding assay and in vivo sublocation analysis.We found that Orc12T356D showed reduced DNA binding activity and Orc1-2T356A binds genomic DNA more tightly.These data indicate that Orc1-2 Thr356 phosphorylation negatively regulates the DDR in S.islandicus by affecting the DNA binding of the core regulator.Protein phosphorylation and dephosphorylation is a covalent,reversible modification of amino acids which is catalyzed by protein kinases（PKs）and dephosphorylated by protein phosphatases（PPs）.SiRe₂030 and SiRe₂056 are Ser/Thr PKs that play a master regulatory role in S.islandicus.So,who is responsible for catalyzing the phosphorylation of Orcl-2?To identify potential PKs that modify Orcl-2,we constructed in-frame deletion mutants of sire₂030 and sire₂056.Genetic analysis of the mutants revealed that SiRe₂030 or SiRe₂056 deletion increases the DNA damage tolerance of S.islandicus,but the expression pattern of key DDR genes in Δsire₂056 is more similar to orc1-2T356A.These results suggested that SiRe₂030 and SiRe₂056 may negatively regulate the S.islandicus DDR through different pathways,and SiRe₂056 is more likely to work by regulating the phosphorylation of orc1-2.In vitro phosphorylation assay showed that both SiRe₂056 and SiRe₂030 could phosphorylate Orc1-2,but the activity of SiRe₂030 was much weaker than that of SiRe₂056.Interestingly,we identified that multiple sites of Orc1-2 were phosphorylated by SiRe₂056 in vitro,but Thr356 was not included.The molecular mechanism of how Orc1-2 is phosphorylated needs further study.Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins,which functions as a global regulator in DNA damage-responsive（DDR）expression.As for Orc1 initiators,the DDR regulator harbors an N-terminal AAA+ATPase domain,and a C-terminal wingedhelix（wH）DNA-binding domain,which are also organized in a similar fashion.The ATPase domain contains Walker A and Walker B motifs responsible for ATP binding and ATP hydrolysis,respectively,and an initiator specific motif（ISM）.According to the crystal structures and amino acid sequences of several reported archaeal initiator Orc1 proteins,we performed structural modeling and sequence alignments on Orc1-2.We found that key amino acid residues in these structural domains are conserved in both types of archaeal Orc1 proteins,including K72（Walker A）,E154（Walker B）and G127/L128（ISM）in the ATPase domain,and two pairs of arginine residues R353/R354 and R381/R383 in the wH domain.To functionally characterize the conserved domains in orc1-2,genome editing plasmids were constructed for generating gene truncations at the C-or N-terminus（ΔN or ΔC）and point substitutions of the conserved amino acids in the conserved domains of ATPase,ISM and wH for the S.islandicus orc1-2 gene.Gene editing experiments revealed that all designed mutations were successfully obtained,except for orc1-2E154A,which inactivates the ATP hydrolysis of the Walker B at the AAA+ATPase domain.These results indicated that inactivation of the ATP hydrolysis of orc1-2 could be lethal to this archaeon and the Orc12E154A-driven cell death may not rely on the transduction of any DNA damage signal.However,the wH mutation can suppress the cell lethality phenotype of orc1-2E154A.It indicates that the process of cell death mediated by orc1-2 Walker B mutant depends on DNA binding.Subsequently,the weak promoter ParaS-38 was used to drive the expression of Walker B mutant in S.islandicus cells.It was found that orc1-2E154A could not activate the DDR of S.islandicus,but could mediate cell cycle arrest at relatively low concentration without any DNA damage signal.Genetic analysis of other orc1-2 mutants showed that disruption of each functional domain completely abolished their DDR regulation.To yield an insight into how orc1-2 could interact with DDR promoters,the designed mutants were expressed in E.coli and purified to apparent homogeneity.The purified wildtype orc1-2 and its mutated derivatives were then analyzed for their DNA binding activities.We found that all structural domains of orc1-2 are important for DNA binding of the noninitiator and R381/R383 in the wH domain and ISM are responsible for the specific DNAprotein interaction.Taken together,both orc1-2 AAA+ATPase domain and wH domain play an important role in the regulation of DDR.The wH as a DNA binding domain in orc1-2,while ATPase domain not only becomes a molecular switch through ATP binding and ATP hydrolysis,but also regulates the specific recognition of orc1-2 to DNA through ISM motif.Furthermore,orc1-2 regulation occurs in two distinct steps:（a）a low content of orc1-2 elicits cell cycle arrest,and this regulation is switched on by ATP binding and turned off by ATP hydrolysis,any failure in turning off the regulation leads to growth inhibition and cell death;（b）an elevated level of orc1-2 activates the expression of DDR genes encoding DNA repair proteins.