Promiscuous Target Interactions in themarinerTransposonHimar1
Karen Lipkow, Nicolas Buisine, Ronald Chalmers
2004
Journal of Biological Chemistry
We have previously characterized the early intermediates of mariner transposition. Here we characterize the target interactions that occur later in the reaction. We find that, in contrast to the early transposition intermediates, the strand transfer complex is extremely stable and difficult to disassemble. Transposase is tightly bound to the transposon ends constraining rotation of the DNA at the single strand gaps in the target site flanking the element on either side. We also find that
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... h the cleavage step requires Mg 2؉ or Mn 2؉ as cofactor, the strand transfer step is also supported by Ca 2؉ , suggesting that the structure of the active site changes between cleavage and insertion. Finally, we show that, in contrast to the bacterial cut and paste transposons, mariner target interactions are promiscuous and can take place either before or after cleavage of the flanking DNA. This is similar to the behavior of the V(D)J system, which is believed to be derived from an ancestral eukaryotic transposon. We discuss the implications of promiscuous target interactions for promoting local transposition and whether this is an adaptation to facilitate the invasion of a genome following horizontal transfer to a new host species. Himar1 is a synthetic mariner transposon reconstituted by "mixing and matching" DNA sequences from almost identical elements in the horn fly, Hematobia irritans, and the lacewing, Chrysoperla plorabunda (1, 2). mariner is a Class II DNA transposon that belongs to a superfamily of elements that includes Tc1 in eukaryotes and the more distantly related IS630-like 1 elements in bacteria (3, 4). mariner elements are extremely widespread in nature, but the vast majority are inactive due to large numbers of point mutations and deletions. The only active examples identified in insects to date are the Mos1 elements from Drosophila mauritiana and Drosophila simulans and a newly discovered mariner from an earwig (5). DNA transposons are well adapted to bacterial hosts and may persist in the genome for an indefinite period of time. In contrast, DNA transposons have a short life span in eukaryotic species and tend to accumulate inactivating mutations during the invasion of a genome (see Ref. 6 and references therein). Therefore, for survival, DNA transposons rely on frequent horizontal transfer between species. As a result of this unusual lifestyle, the presence or absence of mariner does not correlate with the established phylogeny of closely related host organisms. Conversely, almost identical mariner elements are found in more distantly related species (Refs. 1, 6, and 7 and references therein). mariner elements are very compact and contain a single transposase gene, usually flanked by simple terminal-inverted repeats (for review see Ref. 7). They transpose in the germ line and/or soma via a "cut and paste" DNA intermediate and duplicate a TA dinucleotide upon insertion. All of the classical DNA transposons such as mariner are descended from a common ancestor and have an RNase H-like structural fold in the active site with a characteristic DDE(D) motif. These residues serve to coordinate the catalytic metal ion and are shared by a diverse group of proteins including DNA polymerases, RuvC, the V(D)J recombinase Rag1, and the retroviral integrases. However, there are considerable mechanistic differences, even among the transposases. The structure of mariner transposons most closely resembles that of the bacterial insertion sequences (IS elements). However, there are important differences in the molecular mechanism of transposition between the prokaryotic and eukaryotic elements. For example, one apparently universal feature of the bacterial IS elements is that the first nick during the excision step generates the 3Ј-OH at the end of the transposon that is eventually transferred to the target site by a direct transesterification mechanism (8). In mariner and other eukaryotic elements such as Activator and Tam3, the polarity of the reaction chemistry is reversed and the first nick generates the 5Ј-phosphate at the end of the transposon, for example (9). These and other differences may be attributed to a founder effect in the eukaryotic lineage, but this seems unlikely considering other examples of horizontal transfer. More likely is that the differences between prokaryotic and eukaryotic elements are ancient adaptations favored by the different scale and organization of the respective genomes. We have already characterized the early intermediates of Himar1 transposition and documented further important mechanistic differences to the bacterial IS elements (6). In contrast to the bacterial elements, our results suggested that Himar1 transposase can multimerize and initiate catalysis at a single isolated transposon end. This shows that the architecture of the mariner synaptic complex has more in common with V(D)J recombination, which is probably derived from an ancestral eukaryotic transposon. Here we have gone on to charac-
doi:10.1074/jbc.m408759200
pmid:15333635
fatcat:ztckn6pplvdffn3vbhqsctj4pq