Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal

Fuller, James R.; Rice, Phoebe A.

doi:10.7554/elife.21777

Cited by 27 publications

(23 citation statements)

References 48 publications

(74 reference statements)

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“…Thus, it appears that once the hairpin formation step of the reaction has been reached, there is no need for any further major protein movements. It has been proposed that the strain on bent DNA is released upon strand transfer, thereby preventing reaction reversal ( 30 , 69 ).…”

Section: Discussionmentioning

confidence: 99%

Structural insights into the mechanism of double strand break formation by Hermes, a hAT family eukaryotic DNA transposase

Hickman

Voth

Ewis

et al. 2018

Nucleic Acids Research

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Some DNA transposons relocate from one genomic location to another using a mechanism that involves generating double-strand breaks at their transposon ends by forming hairpins on flanking DNA. The same double-strand break mode is employed by the V(D)J recombinase at signal-end/coding-end junctions during the generation of antibody diversity. How flanking hairpins are formed during DNA transposition has remained elusive. Here, we describe several co-crystal structures of the Hermes transposase bound to DNA that mimics the reaction step immediately prior to hairpin formation. Our results reveal a large DNA conformational change between the initial cleavage step and subsequent hairpin formation that changes which strand is acted upon by a single active site. We observed that two factors affect the conformational change: the complement of divalent metal ions bound by the catalytically essential DDE residues, and the identity of the –2 flanking base pair. Our data also provides a mechanistic link between the efficiency of hairpin formation (an A:T basepair is favored at the –2 position) and Hermes' strong target site preference. Furthermore, we have established that the histidine residue within a conserved C/DxxH motif present in many transposase families interacts directly with the scissile phosphate, suggesting a crucial role in catalysis.

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Section: Discussionmentioning

confidence: 99%

Structural insights into the mechanism of double strand break formation by Hermes, a hAT family eukaryotic DNA transposase

Hickman

Voth

Ewis

et al. 2018

Nucleic Acids Research

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“…These hairpin insertion sites should be easily predicted using software tools but an ssDNA probability score (sscount) calculated by mfold software (23) does not correlate with the most prominent insertion sites (Supplementary Figure S1). One possible explanation might be that MuA exhibits increased activity on mismatched targets (40) and the extent to which mismatches are tolerated has yet not been investigated. However, when we searched for mismatched hairpins, no correlations to the read start hot spots were apparent.…”

Section: Discussionmentioning

confidence: 99%

Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposase-based rapid protocol

Radukic

Brandt

Haak

et al. 2019

Preprint

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Monitoring DNA integrity and DNA contaminants in adeno-associated virus (AAV) gene therapy vectors is of major interest, because of clinical applications with increasing therapeutic doses.We here report direct, amplification-free nanopore sequencing of single-stranded AAV DNA using a rapid transposase-based protocol. Direct sequencing of bacteriophage M13 single-stranded DNA supports the finding that single-stranded DNA in general is amenable to direct transposasebased library generation, albeit with increased insertion bias. Sequencing AAV DNA from purified viral particles readily covered the otherwise notoriously difficult to sequence inverted terminal repeats and revealed single-nucleotide variants across the transgene cassette. Significant methylation of the packaged DNA was not identified. Furthermore, nanopore sequencing provided long reads up to full genome coverage and enabled detection of a priori unknown packaged DNA, which sets it apart from short read techniques or qPCR. Long reads directly revealed packaging of two fused genomes and fusions of a genome to the plasmid backbone. Preferred packaging of distinct forms of backbone DNA from producer plasmids, caused by a so far unknown mechanism, were uncovered. The findings promote direct nanopore sequencing as a fast and versatile platform for AAV vector characterization in research and clinical settings even on singlestranded DNA viruses.

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“…However, in vivo the target DNA would not be naked. As MuA greatly prefers prebent target DNA ( 50 ), the multitude of DNA binding proteins found in vivo could have a wide range of effects on the efficiency of target capture by the transpososome. The genomic integration assay also differs from the others in requiring electroporation of functional transpososomes into E. coli .…”

Section: Discussionmentioning

confidence: 99%

Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering

Rasila

Pulkkinen

Kiljunen

et al. 2017

Nucleic Acids Research

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The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA–DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein–protein and protein–DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.

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Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal

Cited by 27 publications

References 48 publications

Structural insights into the mechanism of double strand break formation by Hermes, a hAT family eukaryotic DNA transposase

Structural insights into the mechanism of double strand break formation by Hermes, a hAT family eukaryotic DNA transposase

Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposase-based rapid protocol

Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering

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