Mouse anti-Human SETMAR monoclonal antibody

Mariner transposition is a complex reaction that involves three recombination sites and six strand breaking and joining reactions. This requires precise spatial and temporal coordination between the different components to ensure a productive outcome and minimize genomic instability. We have investigated how the cleavage events are orchestrated within the mariner transpososome. We find that cleavage of the non-transferred strand is completed at both transposon ends before the transferred strand is cleaved at either end. By introducing transposon-end mutations that interfere with cleavage, but leave transpososome assembly unaffected, we demonstrate that a structural transition preceding transferred strand cleavage is coordinated between the two halves of the transpososome. Since mariner lacks the DNA hairpin intermediate, this transition probably reflects a reorganization of the transpososome to allow the access of different monomers onto the second pair of strands, or the relocation of the DNA within the same active site between two successive hydrolysis events. Communication between transposase subunits also provides a failsafe mechanism that restricts the generation of potentially deleterious double-strand breaks at isolated sites. Finally, we identify transposase mutants that reveal that the conserved WVPHEL motif provides a structural determinant of the coordination mechanism.

The RNase H structural fold defines a large family of nucleic acid metabolizing enzymes that catalyze phosphoryl transfer reactions using two divalent metal ions in the active site. Almost all of these reactions involve only one strand of the nucleic acid substrates. In contrast, cut-and-paste transposases cleave two DNA strands of opposite polarity, which is usually achieved via an elegant hairpin mechanism. In the mariner transposons, the hairpin intermediate is absent and key aspects of the mechanism by which the transposon ends are cleaved remained unknown. Here, we characterize complexes involved prior to catalysis, which define an asymmetric pathway for transpososome assembly. Using mixtures of wild-type and catalytically inactive transposases, we show that all the catalytic steps of transposition occur within the context of a dimeric transpososome. Crucially, we find that each active site of a transposase dimer is responsible for two hydrolysis and one transesterification reaction at the same transposon end. These results provide the first strong evidence that a DDE/D active site can hydrolyze DNA strands of opposite polarity, a mechanism that has rarely been observed with any type of nuclease.