DNA-measuring Wadjet SMC ATPases restrict smaller circular plasmids by DNA cleavage

Liu, Hon Wing; Roisné-Hamelin, Florian; Beckert, Bertrand; Li, Yan; Myasnikov, Alexander G.; Gruber, Stephan

doi:10.1016/j.molcel.2022.11.015

Cited by 41 publications

(74 citation statements)

References 55 publications

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“…MksG shares the TOPRIM fold of Topo VI. These data are supported by very recent publications of Wadjet systems from Pseudomonas aeruginoasa PA14 and E. coli GF4-3 ( 63 , 76 ). In these publications the corresponding JetD (MksG) was also shown to be an endonuclease.…”

Section: Discussionsupporting

confidence: 81%

The MksG nuclease is the executing part of the bacterial plasmid defense system MksBEFG

Weiss

Giacomelli

Assaya

et al. 2023

Nucleic Acids Research

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Cells are continuously facing the risk of taking up foreign DNA that can compromise genomic integrity. Therefore, bacteria are in a constant arms race with mobile genetic elements such as phages, transposons and plasmids. They have developed several active strategies against invading DNA molecules that can be seen as a bacterial ‘innate immune system’. Here, we investigated the molecular arrangement of the Corynebacterium glutamicum MksBEFG complex, which is homologous to the MukBEF condensin system. We show here that MksG is a nuclease that degrades plasmid DNA. The crystal structure of MksG revealed a dimeric assembly through its C-terminal domain that is homologous to the TOPRIM domain of the topoisomerase II family of enzymes and contains the corresponding ion binding site essential for DNA cleavage in topoisomerases. The MksBEF subunits exhibit an ATPase cycle in vitro and we reason that this reaction cycle, in combination with the nuclease activity provided by MksG, allows for processive degradation of invading plasmids. Super-resolution localization microscopy revealed that the Mks system is spatially regulated via the polar scaffold protein DivIVA. Introduction of plasmids results in an increase in DNA bound MksG, indicating an activation of the system in vivo.

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Section: Discussionsupporting

confidence: 81%

The MksG nuclease is the executing part of the bacterial plasmid defense system MksBEFG

Weiss

Giacomelli

Assaya

et al. 2023

Nucleic Acids Research

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“…The spatial distribution of anti-phage systems on Streptomyces linear chromosomes could also provide hints on their mechanisms. Strikingly, the two systems that are the most frequently found at the extremities of the chromosomes (Lamassu and Wadjet) both encompass SMC domain, have anti-plasmid activity, and have been proposed to sense the structure of plasmid DNA [34,62,63]. This suggests that the spatial distribution of defence systems might have links with their molecular mechanisms.…”

Section: Streptomyces Linear Chromosomes As a Model To Explore Molecu...mentioning

confidence: 99%

Genomic characterization of the antiviral arsenal of Actinobacteria

et al. 2023

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Phages are ubiquitous in nature, and bacteria with very different genomics, metabolisms, and lifestyles are subjected to their predation. Yet, the defence systems that allow bacteria to resist their phages have rarely been explored experimentally outside a very limited number of model organisms. Actinobacteria (Actinomycetota) are a phylum of GC-rich Gram-positive bacteria, which often produce an important diversity of secondary metabolites. Despite being ubiquitous in a wide range of environments, from soil to fresh and sea water but also the gut microbiome, relatively little is known about the anti-phage arsenal of Actinobacteria. In this work, we used DefenseFinder to systematically detect 131 anti-phage defence systems in 22803 fully sequenced prokaryotic genomes, among which are 2253 Actinobacteria of more than 700 species. We show that, like other bacteria, Actinobacteria encode many diverse anti-phage systems that are often encoded on mobile genetic elements. We further demonstrate that most detected defence systems are absent or rarer in Actinobacteria than in other bacteria, while a few rare systems are enriched (notably gp29-gp30 and Wadjet). We characterize the spatial distribution of anti-phage systems on Streptomyces chromosomes and show that some defence systems (e.g. RM systems) tend to be encoded in the core region, while others (e.g. Lamassu and Wadjet) are enriched towards the extremities. Overall, our results suggest that Actinobacteria might be a source of novel anti-phage systems and provide clues to characterize mechanistic aspects of known anti-phage systems.

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“…Initially, we excluded the nuclease subunit JetD and incubated JetABC with plasmid DNA (pDonor, 1.8 kb) for 10 minutes at room temperature in the presence of ATP prior to grid freezing. This is expected to provide sufficient time for DNA sensing by JET and its priming for cleavage (given that JetABC concentration, rather than JetD, is rate-limiting for cleavage 8 ). This approach yielded a structure of the dimeric core of plasmid-borne JetABC, with an overall resolution of 4.8 Å (Figures 3A and S5; Table S1, Methods).…”

Section: Cryo-em Structure Of Plasmid-bound Jetabcmentioning

confidence: 99%

“…JET systems exhibit a specific capacity to restrict smaller (<80-100 kb) circular DNA molecules through DNA cleavage, independently of DNA sequence and helical topology (Figure 1A) 8,9 . This requires ATP, functional JetC ATPase, and JetD nuclease, giving rise to linear products with some variability in the nature of the DNA ends 6,8,9 . The precise mechanism underlying plasmid restriction by the JET complex remains elusive.…”

Section: Introductionmentioning

confidence: 99%

Structural basis for plasmid restriction by SMC JET nuclease

Roisné-Hamelin,

Liu,

Taschner

et al. 2023

Preprint

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SummaryDNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of circular plasmids through DNA cleavage; yet the mechanisms for target recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage implying that residual unextruded DNA is cleaved. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as principle for non-self DNA recognition and molecular signature for plasmid cleavage. We elucidate key elements of SMC loop extrusion including motor directionality and the structure of a DNA-holding state.

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DNA-measuring Wadjet SMC ATPases restrict smaller circular plasmids by DNA cleavage

Cited by 41 publications

References 55 publications

The MksG nuclease is the executing part of the bacterial plasmid defense system MksBEFG

The MksG nuclease is the executing part of the bacterial plasmid defense system MksBEFG

Genomic characterization of the antiviral arsenal of Actinobacteria

Structural basis for plasmid restriction by SMC JET nuclease

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