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

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.

Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration

“…We first determined the minimal transposon sequences needed for robust activity and validated the importance of each transposase binding site (TBS) found within both left and right ends. Interestingly, our data revealed a broad degree of tolerance to mutagenesis of individual TBSs, a feature corroborated by recent TnsB transposase-DNA structures that show interactions mainly with the DNA backbone rather than specific nucleobases (37–39). The presence of multiple binding sites within each transposon end might allow for accumulative specificity and affinity, and likely play a role in regulating transposition frequency.…”

“…This dataset revealed that individual TBS point mutations can affect efficiency, particularly for positions 1, 6-9, and 12-14, but are not critical for integration. This more lenient sequence requirement is in line with recently published cryo-EM structures of DNA-bound TnsB from Tn 7 and Type V-K CAST systems, which revealed that many protein-DNA interactions occur with the phosphodiester backbone rather than specific nucleobases (37–39).…”