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bioRxiv · 10.1101/2022.08.05.502904

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

Tenjo Castano, F.·Sofos, N.·Lopez-Mendez, B.·fuglsang, a.·Stutzke, L.·Stella, S.·Montoya, G.
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Abstract

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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Tenjo Castano, F., Sofos, N., Lopez-Mendez, B., fuglsang, a., Stutzke, L., Stella, S., Montoya, G.. 2022-08-05. Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon. https://doi.org/10.1101/2022.08.05.502904

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