Molecular mechanisms of replicative transposition
Our research is aimed at a mechanistic understating of the biological activity of macromolecular nucleoprotein complexes using advanced approaches of structural biology (X-ray crystallography and cryogenic electron microscopy) but also a spectrum of complementary biophysical and biochemical methods (small-angle X-ray scattering, analytical ultracentrifugation hydrogen-deuterium exchanges, crosslinking, fluorescence spectroscopy, isothermal titration calorimetry). Currently, we are focused on the genomic rearrangements that accompany the movement of DNA transposons that reside within the host genome as selfish mobile DNA elements and use a self-coded protein, transposase, to move around (or copy itself) without RNA intermediates. They could be found almost in all living organisms and they are a major evolutionary genome shaping force, potent source of genome diversity and instability. Their remnants often form a vast amount of genomic space and have contributed to the evolution in many ways. Their movement, transposase catalysed process of consecutive DNA cleavages and rejoinings, not only disrupts and reorders DNA but it can also provide regulatory sequences that enable the establishment of novel expression patterns and networks. In particular, we are interested in such DNA transposons that use a replicative way of propagation meaning that they do not only jump from one site to the another but they generate new copies progressively increasing their numbers. This has serious consequences mainly in bacteria because these elements are often associated with resistance genes against antibiotics. However, they are also widespread in eukaryotic genomes (Helitrons) and affected evolution of many species.
Projects
Copy-out/paste-in transpostion in ISCth4
Copy-out/paste-in is a replicative transposition pathway used by a majority of families of bacterial insertion sequences, the simplest autonomous DNA transposons. It is distinctive from the other transposition pathways by its two-step mechanism when the first step results in a branched intermediate, “figure-eight”, that includes single stranded DNA circle. The formation of this intermediate is asymmetric meaning that the transposase does not catalyze the same reaction at both ends of the transposon but instead recognizes one end as a target for integration in a sequential manner. This is unusual because in many other cases it has been shown that transposases form highly symmetrical complexes. On the other hand, the second step is currently elusive but it seem to be symmetric. Here, we used the transposase from ISCth4 transposon as a keyhole to provide first insight into the mechanism of asymmetric formation of circular intermediate.
Kosek D., Hickman AB., Ghirlando R., He S, Dyda, F. (2021) Structures of ISCth4 transpososomes reveal the role of asymmetry in copy-out/paste-in DNA transposition. EMBO J., 40, e105666.