The FHA domain protein ArnA functions as a global DNA damage response repressor in the hyperthermophilic archaeon Saccharolobus islandicus

Abstract

Forkhead-associated (FHA) domain proteins specifically recognize phosphorylated threonine via the FHA domain and are involved in signal transduction in various processes especially DNA damage response (DDR) and cell cycle regulation in eukaryotes. Although FHA domain proteins are found in prokaryotes, archaea, and bacteria, their functions are far less clear as compared to the eukaryotic counterparts, and it has not been studied whether archaeal FHA proteins play a role in DDR. Here, we have characterized an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) by genetic, biochemical, and transcriptomic approaches. We find that Δ SisarnA exhibits higher resistance to DNA damage agent 4-nitroquinoline 1-oxide (NQO). The transcription of ups genes, encoding the proteins for pili-mediated cell aggregation and cell survival after DDR, is elevated in Δ SisarnA . The interactions of SisArnA with two predicted partners, SisvWA1 (SisArnB) and SisvWA2 (designated as SisArnE), were enhanced by phosphorylation in vitro . Δ SisarnB displays higher resistance to NQO than the wild type. In addition, the interaction between SisArnA and SisArnB, which is reduced in the NQO-treated cells, is indispensable for DNA binding in vitro . These indicate that SisArnA and SisArnB work together to inhibit the expression of ups genes in vivo . Interestingly, Δ SisarnE is more sensitive to NQO than the wild type, and the interaction between SisArnA and SisArnE is strengthened after NQO treatment, suggesting a positive role of SisArnE in DDR. Finally, transcriptomic analysis reveals that SisArnA represses a number of genes, implying that archaea apply the FHA/phospho-peptide recognition module for extensive transcriptional regulation. IMPORTANCE Cellular adaption to diverse environmental stresses requires a signal sensor and transducer for cell survival. Protein phosphorylation and its recognition by forkhead-associated (FHA) domain proteins are widely used for signal transduction in eukaryotes. Although FHA proteins exist in archaea and bacteria, investigation of their functions, especially those in DNA damage response (DDR), is limited. Therefore, the evolution and functional conservation of FHA proteins in the three domains of life is still a mystery. Here, we find that an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) represses the transcription of pili genes together with its phosphorylated partner SisArnB. SisArnA derepression facilitates DNA exchange and repair in the presence of DNA damage. The fact that more genes including a dozen of those involved in DDR are found to be regulated by SisArnA implies that the FHA/phosphorylation module may serve as an important signal transduction pathway for transcriptional regulation in archaeal DDR.

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

“…This archaeon has been a model for the investigation of antiviral mechanisms by different CRISPR–Cas systems, including I‐A and III‐B subtypes that employ ribonucleoprotein complexes of multiple subunits to mediate nucleic acid interference in a small RNA‐guided fashion 27 , 28 , 29 . In archaea biology research, studies with this crenarchaeon have contributed to the understanding of novel DNA damage‐responsive regulation networks 30 , 31 , 32 , the archaeal cell division system 33 , 34 , and cell cycle regulation 35 , 36 . Versatile genetic tools have been developed for this model, including conventional and novel schemes of genetic manipulations 23 , 37 , highly efficient expression systems 38 , 39 , and CRISPR‐based mutagenesis and gene silencing 40 , 41 , 42 .…”