Structural and Functional Analysis of DndE Involved in DNA Phosphorothioation in the Haloalkaliphilic Archaea Natronorubrum bangense JCM10635

He, Wei; Gao, Haiyan; Wu, Dan; Jiang, Shu‐Heng; Huang, Wanqiu; Chen, Chao; Deng, Zixin; Xiong, Lei; Wu, Geng; Wang, Lianrong

doi:10.1128/mbio.00716-22

Cited by 4 publications

(3 citation statements)

References 33 publications

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“…The predicted N-terminal domain structure also aligns with the DndE protein involved in the phosphorothioation of bacterial DNA in a newly discovered bacterial defense system ( 53 , 54 , 64 , 65 ). Putative nickase activity of supercoiled plasmid was observed with recombinant DndE protein ( 53 ), suggesting the tantalizing possibility of a similar nickase activity harbored in the N-terminal domain of CasDinG. However, the amino acid sequences of DndE and CasDinG N-terminal domain share little conservation (Figure 6 .…”

Section: Discussionmentioning

confidence: 70%

“…A Dali search with the predicted N-terminal domain structure revealed similarities to several nucleic acid binding proteins including bacteriophage encoded transcription regulators ( 49–51 ), the dsDNA binding VirC protein involved in DNA transfer into plants from Agrobacterium ( 52 ), and bacterial defense proteins such as DndE from the DNA phosphorothioate defense system ( 53 , 54 ) and WYL1 of the CRISPR VI-D system ( 55 ). The CasDinG N-terminal domain aligned with highest Z scores to several structures of a P22 phage transcriptional regulator called the ARC repressor.…”

Section: Resultsmentioning

confidence: 99%

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CasDinG is a 5′-3′ dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity

Domgaard,

Cahoon,

Armbrust

et al. 2023

Nucleic Acids Research

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CRISPR-associated DinG protein (CasDinG) is essential to type IV-A CRISPR function. Here, we demonstrate that CasDinG from Pseudomonas aeruginosa strain 83 is an ATP-dependent 5′-3′ DNA translocase that unwinds double-stranded (ds)DNA and RNA/DNA hybrids. The crystal structure of CasDinG reveals a superfamily 2 helicase core of two RecA-like domains with three accessory domains (N-terminal, arch, and vestigial FeS). To examine the in vivo function of these domains, we identified the preferred PAM sequence for the type IV-A system (5′-GNAWN-3′ on the 5′-side of the target) with a plasmid library and performed plasmid clearance assays with domain deletion mutants. Plasmid clearance assays demonstrated that all three domains are essential for type IV-A immunity. Protein expression and biochemical assays suggested the vFeS domain is needed for protein stability and the arch for helicase activity. However, deletion of the N-terminal domain did not impair ATPase, ssDNA binding, or helicase activities, indicating a role distinct from canonical helicase activities that structure prediction tools suggest involves interaction with dsDNA. This work demonstrates CasDinG helicase activity is essential for type IV-A CRISPR immunity as well as the yet undetermined activity of the CasDinG N-terminal domain.

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Section: Discussionmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 99%

CasDinG is a 5′-3′ dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity

Domgaard,

Cahoon,

Armbrust

et al. 2023

Nucleic Acids Research

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show abstract

“…Due to our analysis that predicted tgvAB belongs to the GmrSD/DndB/SspE family, all of which specifically digest phage DNA with unique modifications to 5-hydroxymethylctosine (5hmC)(26, 28, 43), we hypothesized that the T2 tvgAB resistant mutants had non-functional mutations in genes that were associated with unique modifications to 5hmC. We focused on the T2 gene agt , which encodes an α-glucosyltransferase that glucosylates 5hmC bases, due to prior studies showing the homologous GmrSD targets glucosylated phage DNA with these modifications and agt modifies 70% of the cytosines of T4 during an infection(28, 36).…”

Section: Resultsmentioning

confidence: 99%

AVibrio choleraeType IV restriction system targets glucosylated 5-hydroxyl methyl cytosine to protect against phage infection

Gomez,

Waters

2024

Preprint

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A major challenge faced by Vibrio cholerae is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, V. cholerae has evolved a myriad of phage defense systems. Although several novel defense systems have been discovered, we hypothesized more were encoded in V. cholerae given the relative paucity of phage that have been isolated which infect this species. Using a V. cholerae genomic library, we identified a Type IV restriction system consisting of two genes within a 16kB region of the Vibrio pathogenicity island-2 that we name TgvA and TgvB (Type I-embedded gmrSD-like system of VPI-2). We show that both TgvA and TgvB are required for defense against T2, T4, and T6 by targeting glucosylated 5-hydroxymethylcytosine (5hmC). T2 or T4 phages that lose the glucose modification are resistant to TgvAB defense but exhibit a significant evolutionary tradeoff becoming susceptible to other Type IV restriction systems that target unglucosylated 5hmC. We show that additional phage defense genes are encoded in VPI-2 that protect against other phage like T3, secΦ18, secΦ27 and λ. Our study uncovers a novel Type IV restriction system in V. cholerae, increasing our understanding of the evolution and ecology of V. cholerae while highlighting the evolutionary interplay between restriction systems and phage genome modification.

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Reappraisal of the DNA phosphorothioate modification machinery: uncovering neglected functional modalities and identification of new counter-invader defense systems

Rakesh,

Aravind,

Krishnan

2024

Nucleic Acids Research

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The DndABCDE systems catalysing the unusual phosphorothioate (PT) DNA backbone modification, and the DndFGH systems, which restrict invasive DNA, have enigmatic and paradoxical features. Using comparative genomics and sequence-structure analyses, we show that the DndABCDE module is commonly functionally decoupled from the DndFGH module. However, the modification gene-neighborhoods encode other nucleases, potentially acting as the actual restriction components or suicide effectors limiting propagation of the selfish elements. The modification module's core consists of a coevolving gene-pair encoding the DNA-scanning apparatus – a DndD/CxC-clade ABC ATPase and DndE with two ribbon-helix-helix (MetJ/Arc) DNA-binding domains. Diversification of DndE’s DNA-binding interface suggests a multiplicity of target specificities. Additionally, many systems feature DNA cytosine methylase genes instead of PT modification, indicating the DndDE core can recruit other nucleobase modifications. We show that DndFGH is a distinct counter-invader system with several previously uncharacterized domains, including a nucleotide kinase. These likely trigger its restriction endonuclease domain in response to multiple stimuli, like nucleotides, while blocking protective modifications by invader methylases. Remarkably, different DndH variants contain a HerA/FtsK ATPase domain acquired from multiple sources, including cellular genome-segregation systems and mobile elements. Thus, we uncovered novel HerA/FtsK-dependent defense systems that might intercept invasive DNA during replication, conjugation, or packaging.

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Structural and Functional Analysis of DndE Involved in DNA Phosphorothioation in the Haloalkaliphilic Archaea Natronorubrum bangense JCM10635

Cited by 4 publications

References 33 publications

CasDinG is a 5′-3′ dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity

CasDinG is a 5′-3′ dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity

AVibrio choleraeType IV restriction system targets glucosylated 5-hydroxyl methyl cytosine to protect against phage infection

Reappraisal of the DNA phosphorothioate modification machinery: uncovering neglected functional modalities and identification of new counter-invader defense systems

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