Path of DNA within Mu transpososomes: order, dynamics and topology of Mu end-enhancer interactions
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Using a new method called ‘difference topology’, a complete topological analysis of all the intermediates in the Mu transposition pathway has been carried out. The results give us a first look into the intricate architecture of transpososomes made by the Mu transposase. We show that the three participating DNA sites - L (left end), R (right end) and E (enhancer) - are brought together to make an elaborate 5-noded synapse, wherein E crosses R twice and L once, and L and R cross each other two times. This is the most complex DNA arrangement seen to date within a recombination synapse. The order and dynamics of association of these sites was monitored within a series of transpososomes prior to and during synapsis (LER), engagement (type 0), cleavage (type I) and strand transfer (type II) of Mu ends. In the process, we have discovered a new intermediate ER, which likely initiates the assembly pathway. We have found that the enhancer, previously believed to have exited the transpososome as early as in the type 0 complex, instead remains within the transpososome through completion of transposition. Our results suggest that the enhancer may play additional roles in transposition than previously thought.