Orsolya Barabas

Long INterspersed Element-1 (L1) retrotransposons use activities contained within the L1 open reading frame 2-encoded protein (ORF2p) to mobilize throughout the genome via target-site primed reverse transcription (TPRT). The ORF2p endonuclease domain (EN) cleaves genomic DNA to liberate a 3’-hydroxyl (3’-OH) group that is used by the ORF2p reverse transcriptase domain (RT) to synthesize a cDNA copy of its bound L1 RNA template. L1 also can move by EN-independent retrotransposition (ENi), where a 3’-OH group at genomic DNA lesions, dysfunctional telomeres, or stalled replication forks is proposed to prime L1 reverse transcription in the absence of L1 EN cleavage. We previously reported that ribonucleoprotein (RNP) preparations from cells transfected with a human wild-type (WT) L1 or L1 EN-mutant, but not an L1 RT-mutant, can initiate reverse transcription from a DNA oligonucleotide primer/L1 RNA template complex. The WT and EN-deficient L1 RNP preparations also are associated with a nuclease activity that can process a 3’ end modification that precludes DNA synthesis from an oligonucleotide prior to priming the L1 RT reaction. Here, we purified recombinant full-length WT, L1 EN-, and L1 RT-mutant human L1 ORF2p from insect cells. We report that the WT and L1 EN-mutant, but not the L1 RT-mutant, contain an alternative endonuclease activity (alt-EN). Alt-EN activity also is detected in a bacterially expressed L1 ORF2p protein that lacks the L1 EN and ORF2p cysteine-rich domains and a thermostable group II intron-encoded protein. Processing of diverse modified primers demonstrates endonucleolytic cleavage that is eliminated by mutations in the RT active site. We propose that alt-EN is an evolutionarily conserved activity within the RT fold that promoted ENi retrotransposition of primordial retrotransposons prior to the acquisition of an EN domain.

Eukaryotes have linear DNA, and their telomeres are hotspots for transposons, which in some cases took over telomere maintenance. We identified several families of independently evolved telomeric transposons in linear chromosomes and plasmids of cyanobacteria and Streptomyces . Although these elements have one specific transposon end sequence, with the second boundary being the telomere, we can show that they move using two transposon ends, likely when transiently bridged by the telomere maintenance systems. Mobilization of the element and the associated telomere allows replacement of native telomeres, making the host cell dependent on the new transposon telomere system for genome maintenance. This work indicates how telomeric transposons can promote gene transfer both between and within genomes, substantially influencing the evolutionary dynamics of linear genomes.

Eukaryotes have linear DNA and their telomeres are hotspots for transposons, which in some cases took over telomere maintenance. While many bacteria also have linear chromosomes and plasmids, no transposons were known to target bacterial telomeres. Here we show several families of independently evolved telomeric transposons in cyanobacteria and Streptomyces . While these elements have one specific transposon end sequence with the second boundary being the telomere, we can show they move using two transposon ends. Telomeres are transiently bridged by the telomere maintenance systems, providing a duplex substrate for mobilization of the element and the associated telomere. We identify multiple instances where telomeric transposons have replaced native telomeres, making the host cell dependent on the new telomere system for genome maintenance. This work indicates how telomeric transposons can promote gene transfer both between and within genomes, significantly influencing the evolutionary dynamics of linear genomes.

Prokaryotic Mobile Genetic Elements (MGEs) such as transposons, integrons, phages and plasmids, play important roles in prokaryotic evolution and in the dispersal of cargo functions like antibiotic resistance. However, each of these MGE types is usually annotated and analysed individually, hampering a global understanding of phylogenetic and environmental patterns of MGE dispersal. We thus developed a computational framework that captures diverse MGE types, their cargos and MGE-mediated horizontal transfer events, using recombinases as ubiquitous MGE marker genes and pangenome information for MGE boundary estimation. Applied to ∼84k genomes with habitat annotation, we mapped 2.8 million MGE-specific recombinases to six operational MGE types, which together contain on average 13% of all the genes in a genome. Transposable elements (TEs) dominated across all taxa (∼1.7 million occurrences), outnumbering phages and phage-like elements (<0.4 million). We recorded numerous MGE-mediated horizontal transfer events across diverse phyla and habitats involving all MGE types, disentangled and quantified the extent of hitchhiking of TEs (17%) and integrons (63%) with other MGE categories, and established TEs as dominant carriers of antibiotic resistance genes. We integrated all these findings into a resource (proMGE.embl.de), which should facilitate future studies on the large mobile part of genomes and its horizontal dispersal.

Mobile genetic elements (MGEs) sequester and mobilize antibiotic resistance genes across bacterial genomes. Efficient and reliable identification of such elements is necessary to follow resistance spreading. However, automated tools for MGE identification are missing. Tyrosine recombinase (YR) proteins drive MGE mobilization and could provide markers for MGE detection, but they constitute a diverse family also involved in housekeeping functions. Here, we conducted a comprehensive survey of YRs from bacterial, archaeal, and phage genomes and developed a sequence‐based classification system that dissects the characteristics of MGE‐borne YRs. We revealed that MGE‐related YRs evolved from non‐mobile YRs by acquisition of a regulatory arm‐binding domain that is essential for their mobility function. Based on these results, we further identified numerous unknown MGEs. This work provides a resource for comparative analysis and functional annotation of YRs and aids the development of computational tools for MGE annotation. Additionally, we reveal how YRs adapted to drive gene transfer across species and provide a tool to better characterize antibiotic resistance dissemination.

Phosphoprotein phosphatase‐1 (PP1) is a key player in the regulation of phospho‐serine (pSer) and phospho‐threonine (pThr) dephosphorylation and is involved in a large fraction of cellular signaling pathways. Aberrant activity of PP1 has been linked to many diseases, including cancer and heart failure. Besides a well‐established activity control by regulatory proteins, an inhibitory function for phosphorylation (p) of a Thr residue in the C‐terminal intrinsically disordered tail of PP1 has been demonstrated. The associated phenotype of cell‐cycle arrest was repeatedly proposed to be due to autoinhibition of PP1 through either conformational changes or substrate competition. Here, we use PP1 variants created by mutations and protein semisynthesis to differentiate between these hypotheses. Our data support the hypothesis that pThr exerts its inhibitory function by mediating protein complex formation rather than by a direct mechanism of structural changes or substrate competition.

The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc⁺/int⁻ transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc⁺/int⁻ transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change.