Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element

Abstract

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2, have previously been identified in moso bamboo (Phyllostachys edulis). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR’s affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5–2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2. The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies.

The Dynamism of Transposon Methylation for Plant Development and Stress Adaptation

“…Insufficiency of enzymes encoded by TEs may explain the insufficient quantity for the transposition process. For instance, transposition of Ppmar1 and Ppmar2 (Mariner-like elements (MLEs) isolated from Moso bamboo) is determined by the quantity of transposases present inside the nucleus [156,157]. This suggests that MLEs generally have the potential to develop a self-regulatory strategy that can control their amplification and copy numbers by minimization of transposases.…”

Genetics and genomics of moso bamboo (Phyllostachys edulis): Current status, future challenges, and biotechnological opportunities toward a sustainable bamboo industry

“…Additionally, they identified transposon variations that depict adaptive responses during the evolutionary process of moso bamboo. Subsequently, through functional analyses of transposons, Ramakrishnan, Zhou, Pan, Hänninen, Yrjälä, et al (2019), Ramakrishnan, Zhou, Pan, Hänninen, Tang, et al (2019) identified activities of mariner‐like elements ( MLE ) of the moso bamboo, drawing evolutionary connotations.…”

“…Previous studies attempted to understand the transposition control of MLEs . Ramakrishnan, Zhou, Pan, Hänninen, Yrjälä, et al (2019) were able to alter the transposition frequency of Ppmar2 by 1.5–2.0 times more than that of the wild‐type yeast by increasing the affinities of terminal inverted repeats (TIRs) toward the DNA‐binding domain of TPase. Further, the nuclear export signal (NES) domains of both Ppmar1 and Ppmar2 elements regulate the nuclear export of TPases that ultimately influences the transposition activity of MLEs (Ramakrishnan, Zhou, Pan, Hänninen, Tang, et al, 2019).…”

Artificial optimization of bamboo Ppmar2 transposase and host factors effects on Ppmar2 transposition in yeast

“…Two full-length MLEs named Ppmar1 and Ppmar2 were previously identified from the genome of moso bamboo (Phyllostachys edulis) (Zhou et al, 2015). Ppmar1 and Ppmar2 were shown to be transposable in Arabidopsis thaliana and yeast (Zhou et al, 2016;Zhou et al, 2017;Ramakrishnan et al, 2019a;Ramakrishnan et al, 2019b). Site-directed mutation boosted the activity of the Ppmar1 mutant (S171A) by more than 10-fold (Zhou et al, 2017).…”

“…expression vector, pAG415gal-ccdB, as described by Ramakrishnan et al (2019a;2019b). Ppmar2 non-autonomous transposon (Ppmar2NA) was cloned by amplifying its 5' and 3' TIRs as well as the adjacent sequence, followed by an overlap PCR.…”