Functional characterization of an active Rag-like transposase

Abstract

The formation of diverse immunoglobulin genes results in part from Rag protein-mediated DNA double-strand breaks at the edges of immunoglobulin gene segments, followed by combinatorial reassembly of these segments. We report that a Transib transposase from the insect Helicoverpa zea is active in vitro and that its breakage and joining activities mimic those of Rag, providing strong evidence that Rag and Transib transposases were derived from a common progenitor.

Figure 1. Hztransib transposase can promote coupled cleavage and strand transfer

a. The pathway of transposition. The transposase introduces a double strand break between a transposon end and the flanking donor DNA to expose the 3′ OH transposon end. The joining of one 3′ OH to one target strand a plasmid target generates a nicked circle product (Single End Join; SEJ). The joining of two 3′ OH ends to two target strands at complementary positions in a single target plasmid yields a linearized version of the plasmid attached to the two end oligonucleotides (Double End Join; DEJ). b. Reactions contained wildtype or mutant Hztransib transposase as indicated. We suspect the lower transposase-dependent band likely results from end binding.

Figure 2. Analysis of the Hztransib DNA cleavage mechanism

a. The mechanism of transposition. Transposition begins with a nick at the 5′ end of the transposon end. The newly exposed donor 3′ OH then attacks the complementary strand to form a hairpin and release the transposon end. b. In vitro transposition reactions were carried out using the transposon end fragments labeled as indicated, transposases as indicated and displayed on a denaturing acrylamide gel. The image is of a single gel with a masked lane.

Figure 3. Hztransib Transposition in vitro using pre-cleaved transposon ends

a. Target joining by decreasing amounts of wildtype transposase. The reactions contained 1×, 1/3×, 1/9× and 1/27× transposase. b. Target joining by transposase mutants. The transposon end substrate was a 294 bp Hztransib left end labeled at both 5′ ends (asterisk) and reactions contained wildtype or mutant Hztransib transposase as indicated. The reaction products were displayed on a native agarose gel. c. The 3′ OH transposon end joins to target DNA. The transposon end substrate was a 50 bp Hztransib left end labeled only at the internal 5′ end* (asterisk) of the strand that joins covalently to target DNA.