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DNA Transposition at Work.

ABSTRACT: DNA transposons are defined segments of DNA that are able to move from one genomic location to another. Movement is facilitated by one or more proteins, called the transposase, typically encoded by the mobile element itself. Here, we first provide an overview of the classification of such mobile elements in a variety of organisms. From a mechanistic perspective, we have focused on one particular group of DNA transposons that encode a transposase with a DD(E/D) catalytic domain that is topologically similar to RNase H. For these, a number of three-dimensional structures of transpososomes (transposase-nucleic acid complexes) are available, and we use these to describe the basics of their mechanisms. The DD(E/D) group, in addition to being the largest and most common among all DNA transposases, is the one whose members have been used for a wide variety of genomic applications. Therefore, a second focus of the article is to provide a nonexhaustive overview of transposon applications. Although several non-transposon-based approaches to site-directed genome modifications have emerged in the past decade, transposon-based applications are highly relevant when integration specificity is not sought. In fact, for many applications, the almost-perfect randomness and high frequency of integration make transposon-based approaches indispensable.

SUBMITTER: Hickman AB

PROVIDER: S-EPMC6380494 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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DNA Transposition at Work.

Hickman Alison B AB Dyda Fred F

Chemical reviews 20160517 20

DNA transposons are defined segments of DNA that are able to move from one genomic location to another. Movement is facilitated by one or more proteins, called the transposase, typically encoded by the mobile element itself. Here, we first provide an overview of the classification of such mobile elements in a variety of organisms. From a mechanistic perspective, we have focused on one particular group of DNA transposons that encode a transposase with a DD(E/D) catalytic domain that is topologica ...[more]

PMID: 27187082

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Project description:BACKGROUND:Completed genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA transposition. RESULTS:Here we developed a universal in vivo platform for the analysis of transposition frequency with class II mobile elements, i.e., DNA transposons. For each particular transposon system, cloning of the transposon ends and the cognate transposase gene, in three consecutive steps, generates a multifunctional plasmid, which drives inducible expression of the transposase gene and includes a mobilisable lacZ-containing reporter transposon. The assay scores transposition events as blue microcolonies, papillae, growing within otherwise whitish Escherichia coli colonies on indicator plates. We developed the assay using phage Mu transposition as a test model and validated the platform using various MuA transposase mutants. For further validation and to illustrate universality, we introduced IS903 transposition system components into the assay. The developed assay is adjustable to a desired level of initial transposition via the control of a plasmid-borne E. coli arabinose promoter. In practice, the transposition frequency is modulated by varying the concentration of arabinose or glucose in the growth medium. We show that variable levels of transpositional activity can be analysed, thus enabling straightforward screens for hyper- or hypoactive transposase mutants, regardless of the original wild-type activity level. CONCLUSIONS:The established universal papillation assay platform should be widely applicable to a variety of mobile elements. It can be used for mechanistic studies to dissect transposition and provides a means to screen or scrutinise transposase mutants and genes encoding host factors. In succession, improved versions of transposition systems should yield better tools for molecular biology and offer versatile genome modification vehicles for many types of studies, including gene therapy and stem cell research.

Dataset Information

DNA Transposition at Work.

Publications

DNA Transposition at Work.

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