DNA transposons can be employed for stable gene transfer in vertebrates. The Sleeping Beauty (SB) DNA transposon has been recently adapted for human application and is being evaluated in clinical trials, however its molecular mechanism is not clear. SB transposition is catalyzed by the transposase enzyme, which is a multi-domain protein containing the catalytic and the DNA-binding domains. The DNA-binding domain of SB transposase contains two structurally independent subdomains, PAI and RED. Recently, the structures of the catalytic domain and the PAI subdomain have been determined, however no structural information on the RED subdomain and its interactions with DNA has been available. Here, we used NMR spectroscopy to determine solution structure of RED subdomain and characterize its interactions with the transposon DNA.
Significance statement: The (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon adapted for human gene therapy. Therefore, it is particularly important to have a mechanistic understanding of SB transposase activity. Structural information at the atomic level is vital for such understanding. We report the structure of the last missing piece of SB transposase, the RED subdomain and provide insight into its folding and DNA-binding properties. This article is protected by copyright. All rights reserved.
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Abstract
The solution NMR structure of the isolated thumb subdomain of HIV-1 reverse transcriptase (RT)*has been determined. A detailed comparison of the current structure with dozens of the highest resolution crystal structures of this domain in the context of the full-length enzyme...
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Abstract
The solution NMR structure of the isolated thumb subdomain of HIV-1 reverse transcriptase (RT)Â*has been determined. A detailed comparison of the current structure with dozens of the highest resolution crystal structures of this domain in the context of the full-length enzyme reveals that the overall structures are very similar, with only two regions exhibiting local conformational differences. The C-terminal capping pattern of the αH helix is subtly different, and the loop...
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Abstract
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Abstract
The Sleeping Beauty (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon thus far in clinical trials for human gene therapy. In the absence of atomic level structural information, the development of SB transposon relied primarily on the biochemical and genetic homology data. While these studies were successful and have yielded hyperactive transposases, structural information is needed to gain a mechanistic understanding of transposase activity and...
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ABSTRACT
The Sleeping Beauty (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon thus far in clinical trials for human gene therapy. In the absence of atomic level structural information, the development of SB transposon relied primarily on the biochemical and genetic homology data. While these studies were successful and have yielded hyperactive transposases, structural information is needed to gain a mechanistic understanding of transposase activity and...
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NMR solution structure of the RED subdomain of the Sleeping Beauty transposase
Linear Mode
