A clade of RHH proteins ubiquitous in Sulfolobales and their viruses regulates cell cycle progression

Li, Xuyang; Cristina, Lozano-Madueño; Martínez-Álvarez, Laura; Peng, Xu

doi:10.1093/nar/gkad011

Cited by 4 publications

(2 citation statements)

References 78 publications

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“…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 .…”

Section: Introductionmentioning

confidence: 99%

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

Zhao,

Bi,

Wang

et al. 2024

mLife

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Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools, which include efficient genome editing, gene silencing, and robust protein expression systems. However, plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid. Although this plasmid coexists with pRN1 in its original host, early attempts to test pRN1‐based vectors consistently failed to yield any stable host–vector system for Sa. islandicus. We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon. We identified a putative target sequence in orf904 encoding a putative replicase on pRN1 (target N1). Mutated targets (N1a, N1b, and N1c) were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid interference assay. The results revealed that the original target triggered CRISPR immunity in this archaeon, whereas all three mutated targets did not, indicating that all the designed target mutations evaded host immunity. These mutated targets were then incorporated into orf904 individually, yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed (pN1aSD, pN1bSD, and pN1cSD). Sa. islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors, but pN1cSD lost the capability for replication. These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase. We further showed that pRN1‐based and pRN2‐based vectors were stably maintained in the archaeal cells either alone or in combination, and this yielded a dual plasmid system for genetic study with this important archaeal model.

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Section: Introductionmentioning

confidence: 99%

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

Zhao,

Bi,

Wang

et al. 2024

mLife

View full text Add to dashboard Cite

show abstract

“…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).…”

Section: Introductionmentioning

confidence: 99%

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

Zhao,

Bi,

Wang

et al. 2023

Preprint

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Sulfolobus species belonging to crenarchaea code for a novel DNA damage response (DDR) system in which an Orc1/Cdc6 family protein, Orc1-2, initiates a global cellular response to DNA damage by functioning as a global transcriptional regulator. However, the mechanism that modulates Orc1-2 activity upon DNA damage remains unclear. In this study, we found that Sulfolobus islandicus Orc1-2 was phosphorylated under normal growth and non-phosphorylated upon DNA damage treatment at Thr356. Interestingly, the phospho-mimetic mutant orc1-2T356D exhibit defective DDR processes, while the non-phosphorylatable mutant orc1-2T356A shows prolonged activation of DDR in which the expression of representatives DDR genes are significantly elevated. DNA binding assays revealed that the Orc1-2T356D protein showed reduced DNA binding activity. In contrast, the Orc1-2T356A protein shows enhanced chromosome binding. These data indicate that Orc1-2 phosphorylation negatively regulates the DNA damage response in Sulfolobus by modulating the DNA binding activity of the core regulator.

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Unraveling the structure and function of a novel SegC protein interacting with the SegAB chromosome segregation complex in Archaea

Lin,

Yen,

Shen

et al. 2024

Nucleic Acids Research

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Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5–15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.

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A clade of RHH proteins ubiquitous in Sulfolobales and their viruses regulates cell cycle progression

Cited by 4 publications

References 78 publications

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

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

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

Unraveling the structure and function of a novel SegC protein interacting with the SegAB chromosome segregation complex in Archaea

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