Structural basis of transposon end recognition explains central features of Tn7 transposition systems

Kaczmarska, Zuzanna; Czarnocki-Cieciura, Mariusz; Górecka-Minakowska, Karolina M.; Wingo, Robert J.; Jackiewicz, Justyna; Zajko, Weronika; Poznański, Jarosław; Rawski, Michał; Grant, Timothy; Peters, Joseph E.; Nowotny, Marcin

doi:10.1016/j.molcel.2022.05.005

Cited by 23 publications

(30 citation statements)

References 62 publications

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“…However, two protuberances could be observed associated to an elongated density, suggesting the presence of two shTnsB protomers bound to a dsDNA face of a SR-LTR set. The elongated density assigned to the DNA is bent in a similar manner as in the recently published structure of the RE bound ecTnsB 19 , further supporting that the low-resolution map corresponds to a pre-transposition complex.…”

Section: Resultssupporting

confidence: 83%

“…The ecTnsB end-complex structure has provided information on the recognition of the transposon ends 19 , but so far, there is no structural information on their STC complex. However, the conformation of the ecTnsB subunits in the end complex resemble the arrangement adopted by the DDE domain of the shTnsB3 and 4 protomers in the shTnsB-STC complex.…”

Section: Resultsmentioning

confidence: 99%

“…Recent studies have described new CRISPR-Cas systems that associate with Tn 7 like-transposon proteins 17–19 7 . These CAST systems recruit the CRISPR-Cas machinery and “parasitize” on it to direct DNA integration at a specific locus 4 .…”

Section: Discussionmentioning

confidence: 99%

“…1). The structure of E. coli (ec) TnsB in complex with the end of the transposon has provided new evidence explaining differences in the recognition of the left and right ends of the element 19 . However, the sequence identity of ecTnsB with shTnsB is low, thus making it difficult to understand the details of the integration mechanism of Tn7/CASTs and other transposon systems containing integrases of the DDE family.…”

Section: Introductionmentioning

confidence: 99%

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Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon

Castano

Sofos

López-Méndez

et al. 2022

Preprint

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CRISPR-associated transposons (CASTs) represent unique mobile genetic elements that co-opted CRISPR-Cas immune systems for RNA-guided DNA transposition. Type V-K CAST is composed by Cas12k, TniQ, TnsC and TnsB. Here, we present the 2.46 A cryoelectron microscopy structure of the Scytonema hofmannii CAST TnsB transposase in complex with the strand transfer DNA in a post-catalytic state. The shTnsB strand transfer complex maintains the intertwined architecture of the MuA phage transpososome. However, the building of the assembly depends on different local interactions. The protein-DNA complex forms a pseudo-symmetrical assembly in which the 4 protomers of shTnsB adopt two different conformations. The recognition of the transposon ends is accomplished by two small helical domains. The two protomers involved in the strand transfer reaction display a catalytically competent active site composed by three acidic residues (DDE), while the other two, which play a key role in the complex architecture, show catalytic pockets where the DDE residues are not properly positioned for cleavage. Quantification of in vivo transposition assays of mutants in key DNA binding residues, reveals that the lack of specificity generally decreases activity, but it could increase transposition in some cases. Our structure sheds light on the strand transfer reaction of the DDE DNA transposases and offers new insights into RNA-guided transposition in CAST systems.

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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon

Castano

Sofos

López-Méndez

et al. 2022

Preprint

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“…However, modeling the structure of this complex by superposing an ADP-bound TnsC hexamer onto the Cas12k-TnsC transposon recruitment complex suggests that the insertion site would be located approximately 28-34 bp from the edge of the TnsC hexamer. It is possible that the discrepancy might be due to the large physical footprint of the TnsB-transposon complex, as indicated by a recent structural study of the E. coli Tn7 TnsB bound to transposon end DNA (Kaczmarska et al, 2022). The molecular ruler mechanism determining the distance between the Cas12k target and transposon insertion sites thus remains unclear and awaits further investigation.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for RNA-mediated assembly of type V CRISPR-associated transposons

Schmitz

Querques

Oberli

et al. 2022

Preprint

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CRISPR systems have been co-opted by Tn7-like elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprising RNA-guided Cas12k, TniQ, TnsC and, unexpectedly, the ribosomal protein S15. Complex assembly on target DNA results in complete R-loop formation mediated by critical interactions between TniQ and the trans-activating crRNA, and is coupled with TniQ-dependent nucleation of a TnsC filament. In vivo transposition assays corroborate our structural findings, and biochemical and functional analyses of S15 supports its role as a bona fide component of the type V crRNA-guided transposition machinery. Altogether, our work uncovers key aspects of the mechanisms underpinning RNA-mediated assembly of CRISPR-associated transposons that will guide their development as programmable site- specific gene insertion tools.

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Structural basis for the assembly of the type V CRISPR-associated transposon complex

Schmitz¹,

Querques²,

Oberli³

et al. 2022

Cell

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Structural basis of transposon end recognition explains central features of Tn7 transposition systems

Cited by 23 publications

References 62 publications

Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon

Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon

Structural basis for RNA-mediated assembly of type V CRISPR-associated transposons

Structural basis for the assembly of the type V CRISPR-associated transposon complex

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