Oger, C. A. | bioRxiv Science

bioRxiv2026

A replication hijacking mechanism for Tn3-family replicative transposition

Transposition of all classes of transposable elements generates DNA intermediates that must be processed by the host to be effective. However, the mechanisms whereby transposons communicate with cellular DNA-processing machineries remain poorly investigated. Here, we provide convergent genetic and biochemical evidence that replicative transposition of the Tn3-family transposon Tn4430 is strictly coupled to replication of the target. Blocking target replication abolishes transposition, while blocking replication of the transposon donor molecule has no effect. Furthermore, the insertion preference of Tn4430 was found to be altered by the direction of replication and potential replication impediments within the target, suggesting a functional link between the integration mechanism and replication fork progression. In vitro, the transposase TnpA was found to specifically bind to fork-like DNA structures that mimic replication intermediates. Compared to linear DNA fragments, these structures are efficient substrates for TnpA-catalysed end joining. Strand transfer occurred immediately downstream of the fork, poising the transposon for replication. Together, the data suggest a mechanism in which the transposon targets DNA replication intermediates to directly recruit the host replication machinery at the time of transposition. This "replication hijacking" mechanism contrasts with classical "replication hiring" mechanisms during which replication is recruited after strand transfer. SIGNIFICANCE STATEMENTBacterial transposons of the Tn3 family constitute a threat for human health, being continuously involved in the emergence and spread of new antimicrobial resistances amongst pathogens. The success of these elements is based on their replicative mode of transposition allowing them to produce a new copy of themselves whenever they move. Here, we provide evidence that, rather than recruiting the host replication machinery after integration as is proposed in textbook models for replicative transposition, Tn3-family transposons directly transpose into ongoing replication intermediates. We propose that this "replication hijacking" mechanism provides a means of synchronizing transposition with cell replication activity, thus optimizing the movement of transposons with the expansion of bacterial populations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/728999v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@c04da0org.highwire.dtl.DTLVardef@1285b5aorg.highwire.dtl.DTLVardef@859c5corg.highwire.dtl.DTLVardef@1dc9ce8_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

SPO11 dimerization controls meiotic DNA double-strand break formation

SPO11 initiates meiotic recombination through the induction of programmed DNA double-strand breaks (DSBs), but this catalytic activity had never been reconstituted in vitro. Here, using Mus musculus SPO11, we report a biochemical system that recapitulates all the hallmarks of meiotic DSB formation. We show that SPO11 catalyzes break formation in the absence of any partners and remains covalently attached to the 5' broken strands. We find that target site selection by SPO11 is influenced by the sequence, bendability and topology of the DNA substrate, and provide evidence that SPO11 can reseal single-strand DNA breaks. In addition, we show that SPO11 is monomeric in solution and that cleavage requires dimerization to reconstitute two hybrid active sites. SPO11 and its partner TOP6BL form a 1:1 complex that catalyzes DNA cleavage with a similar activity to SPO11 alone. However, the complex binds DNA ends with higher affinity, suggesting a potential role post-cleavage. We propose a model where additional partners of SPO11 required for DSB formation in vivo assemble biomolecular condensates that recruit SPO11-TOP6BL, enabling dimerization and cleavage. Our work establishes SPO11 dimerization as the fundamental mechanism that controls the induction of meiotic DSBs.

biochemistry↗

bioRxiv2022

Metamorphism of catalytic domain controls transposition in Tn3 family transposases

Transposons account for a remarkable diversity of mobile genetic elements that play the role of genome architects in all domains of life. Tn3 is a family of widespread and among first identified bacterial transposons notorious for their contribution to dissemination of antibiotic resistance. Transposition within this family is mediated by a large TnpA transposase facilitating both transposition and target immunity. The structural framework for understanding the mechanism of the TnpA transposition is however absent. Here, we describe the cryo-EM structures of TnpA from Tn4430 in apo form and paired with transposon ends. We show that TnpA has a unique architecture and exhibits a family-specific regulation mechanism involving metamorphic refolding of the RNase H-like catalytic domain. The TnpA structure, constrained by a double dimerization interface, creates a unique topology that suggests a specific role for target DNA in transpososome assembly, activation, and target immunity.

molecular biology↗