Universal platform for quantitative analysis of DNA transposition

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Here we developed a universal <it>in vivo </it>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 <it>lacZ</it>-containing reporter transposon. The assay scores transposition events as blue microcolonies, papillae, growing within otherwise whitish <it>Escherichia coli </it>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 IS<it>903 </it>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 <it>E. coli </it>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.</p> <p>Conclusions</p> <p>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.</p

Flexibility in MuA Transposase Family Protein Structures: Functional Mapping with Scanning Mutagenesis and Sequence Alignment of Protein Homologues

Peer reviewe

ALS and Parkinson's disease genes CHCHD10 and CHCHD2 modify synaptic transcriptomes in human iPSC-derived motor neurons

Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.Peer reviewe

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

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.Peer reviewe

Location of insertions within the amino acid sequence and relative transposition activity of each mutant.

<p>Amino acid positions are numbered under the sequence. Secondary structures (determined with DSSP, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922-Kabsch1" target="_blank">[74]</a>) are indicated under the corresponding sequence as bars (α-helices) or arrows (β-strands). Small vertical arrows point to the exact location of each insertion (in each three-letter amino acid code, the first letter represents the first nucleotide of the corresponding codon, etc.), and the attached coloured boxes indicate ranges of transposition activity of each mutant relative to the wild type activity (colors denoting the percentage ranges are shown at the lower-right). The number within the boxes indicate the observed relative activity.</p

Mu transpososome architecture at post-integration stage and MuA regions allowing pentapeptide insertions.

<p>The overall organization is sketched according to the unpublished crystallographic structure of Mu transpososome (P. Rice and S.P. Montaño, personal communication). The structure constitutes an essential framework for a meaningful interpretation of the functional data (see Discussion). Transposon end segments are shown with black lines, MuA binding sites (R1 and R2) are highlighted with rectangles, and the attached target DNA is shown in magenta. The catalytic MuA protomers are shown in orange and the non-catalytic protomers in brown. Insertion-tolerant subdomains are highlighted with red. The arrows (shown only for one protomer) indicate those linker and loop regions, in which insertions are tolerated well (wild type protein activity retained). The catalytic protomers act <i>in trans</i>, i.e. the protomer bound to one end catalyzes DNA cleavage and joining reactions in the other end.</p

Identification of insertion-tolerant regions in MuA on the basis of pentapeptide insertion analysis and alignment-based data.

<p>Above the amino acid sequence are shown with arrows the pentapeptide-insertion tolerant sites (activity level ≥1%) colour-coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone-0037922-g004" target="_blank">Figure 4</a>. The percentage range chart is shown at the lower-right. Two or three arrows per site are indicative of insertions involving more than one reading frame. Below the amino acid sequence are shown the secondary structural elements (arrows and rectangles). The elements are connected with line segments indicating the length of each PDB structure. Below the structural elements are shown the subdomains as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone-0037922-g003" target="_blank">Figure 3</a>. Above the bolded line, the downward black and upward white arrows represent the alignment-based insertion and deletion (indel) data, respectively (each particular indel precedes the marked amino acid). The maximum indel length at each site is indicated by a number shown above each arrow (data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922.s001" target="_blank">Figure S1</a>). The stars indicate the DDE-motif residues (D269, D336, E392).</p

Assembly and function of Mu transpososome core.

<p>This pathway is based on <i>in vitro</i> studies and utilizes a minimum number of macromolecular components <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922-Savilahti1" target="_blank">[27]</a>. The <i>in vivo</i> assay described earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922-Pajunen3" target="_blank">[60]</a> and used here (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922.s001" target="_blank">Figure S1</a>) is a close mimic of this minimal-component <i>in vitro</i> system with regard the following features: (i) the configuration includes two MuR-ends (with R1 and R2 MuA binding sites), (ii) the phage-encoded MuB protein and (iii) transpositional enhancer are not included. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922.s001" target="_blank">Figure S1</a> for a full description of Mu transposition pathway and its comparison to the pathway used in the papillation analysis. The R1 and R2 MuA binding sites are shown as rectangles. MuA is drawn as a tetramer of yellow circles and target DNA is shown in purple. The small arrows on the target DNA indicate the 5-bp staggered locations for strand transfer on the two strands. The dots in the assembled transpososome indicate the Mu end cleavage sites.</p