Abstract
Cut-and-paste DNA transposons of the mariner/Tc1 family are useful tools for genome engineering and are inserted specifically at TA target sites. A crystal structure of the mariner transposase Mos1 (derived from Drosophila mauritiana), in complex with transposon ends covalently joined to target DNA, portrays the transposition machinery after DNA integration. It reveals severe distortion of target DNA and flipping of the target adenines into extra-helical positions. Fluorescence experiments confirm dynamic base flipping in solution. Transposase residues W159, R186, F187 and K190 stabilise the target DNA distortions and are required for efficient transposon integration and transposition in vitro. Transposase recognises the flipped target adenines via base-specific interactions with backbone atoms, offering a molecular basis for TA target sequence selection. Our results will provide a template for re-designing mariner/Tc1 transposases with modified target specificities.
Highlights
Deoxyribonucleic acid (DNA) forms the basis of all known living organisms
Transposases are enzymes involved in the movement of sections of DNA within the genome
Understanding the complex mechanism by which the transposase can remove and insert a section of DNA would allow these enzymes to be used as biomolecular tools
Summary
Despite the fundamental importance of deoxyribonucleic acid (DNA) to our existence, our knowledge of DNA and its functional behaviour remains limited. Fluorescence techniques are ideally suited for probing the conformational dynamics within DNA, due to their inherent sensitivity and selectivity, and the noninvasiveness of optical excitation. Both steady-state and time-resolved fluorescence have been utilised in this thesis. As many important processes in DNA occur on timescales that are too short to be observed by common time-resolved fluorescence measurements, there is a necessity for the development of ultrafast fluorescence spectroscopic techniques that are suited to the study of biological molecules in the ultraviolet (UV) spectral region.
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