Dissection of Functional Domains of Orc1-2, the Archaeal Global DNA Damage-Responsive Regulator

Abstract

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 AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, 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) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope

“…Next, we asked whether strong TAMs can be used for SNE editing by reducing the expression level of the effector complex. To this end, we replaced the original P araS promoter driving the expression of the TnpB and reRNA with P araS -m38 59 , a mutated version with ca. 1/8 strength of the original one (Supplementary Fig.…”

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 .…”

Rationale design of unrestricted pRN1 derivatives and their application in construction of a dual plasmid vector system forSaccharolobus islandicus

“…This archaeon has been a model for 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 (23, 24, 25). In archaea biology research, studies with this crenarchaeon have contributed to the understanding of novel DNA damage-responsive regulation networks (26, 27, 28), the archaeal ESCRT cell division system (29, 30) and cell cycle regulation (31, 32). At present, versatile genetic tools have already been developed for this model, including conventional and novel schemes of genetic manipulations (19, 33), highly efficient expression systems (34, 35) and CRISPR-based mutagenesis and gene silencing (36, 37, 38).…”