Abstract
Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB's applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases.
Highlights
Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms
In the Mos[1] paired-end complex (PEC), the clamp loop is extended and interacts with the extended linker and both tnp ends across the dimer interface, playing an important role in PEC assembly[7]
The most surprising feature of the SB100X catalytic domain structure concerns the clamp loop, which is inserted within the RNaseH-fold of Tc1/mariner transposases
Summary
Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. Rational engineering of novel SB variants is currently hampered by the lack of specific mechanistic and structural data, for the transposase catalytic domain To overcome this limitation, here we solve the first crystal structure of the SB catalytic domain and, based on this, generate a model of the SB transpososome containing transposon end as well as target DNA. Our work provides novel insights into SB’s mechanism and demonstrates how structure-based design can help generate further designer transposases
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
