Mechanistic Insights into TnsP : The Candidate Target Selector of the LoPAST Transposon

Diplôme Master Mechanistic Insights into TnsP : The Candidate Target Selector of the LoPAST Transposon eng 2025-12-18 author Kasri Fatma Zohra 966749 director Barabas Orsolya 938832 Département de biologie moléculaire et cellulaire (MOCEL) author Fülöp Máté 952173 Département de biologie moléculaire et cellulaire (MOCEL) 54 Telomeric transposons are a newly identified class of mobile genetic elements that integrate into the termini of linear bacterial chromosomes, thereby acting as “mobile telomeres”. In cyanobacterial PAST elements, the small accessory protein TnsP is proposed to serve as the target site selector for transposition, yet its DNA binding mechanism has remained unclear. Using electrophoretic mobility shift assays (EMSA), we demonstrate that TnsP binds efficiently to linear DNA in vitro but shows no detectable interaction with either supercoiled or topoisomerase I–relaxed circular plasmids, indicating that DNA linearity, not supercoiling, is the primary determinant for binding. The binding is DNA length-dependent and occurs regardless of end structure, as both blunt-ended and sticky-ended linear substrates are recognized equally. Systematic analysis with duplexes ranging from 9 to 35 bp reveals a minimal binding footprint of >11 bp, with clear complex formation beginning at 13 bp and higher- order complexes appearing on longer fragments, consistent with sequential loading of multiple TnsP dimers. Quantitative affinity measurements by microscale thermophoresis (MST) yield dissociation constants of 2.13 ± 0.79 µM for a 13 bp probe and 1.25 ± 1.20 µM for a 27 bp probe, values that are statistically indistinguishable and thus provide limited evidence for cooperative binding under the tested conditions. Together, these results clarify that TnsP functions as a length-dependent DNA binding protein that preferentially engages linear duplex DNA regardless of the end configuration, providing mechanistic insight into how telomeric transposons target chromosome ends for mobility. Dewey 570 Molecular Biosciences, Genetics, Development & Evolution (MBGDE) 7daed2ee-1311-4824-9df9-2e51260a966a Département de biologie moléculaire et cellulaire 16484 false 1347 Groupe Barabas a433150f-9f63-4c18-845f-df8ba1e73e1b Master thesis Restricted Master_thesis_Fatma-Zahra_Kasri.pdf 9308572 application/pdf 1ae56a76134f55b1dedd3315ec194c5b252af850d7c29c367f79a8220e35d6be restrictedAccess Faculté des sciences / Section de biologie / Département de biologie moléculaire et cellulaire

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Kasri, Fatma Zohra

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