Abstract
Antigen receptor gene rearrangements are initiated by the RAG1/2 protein complex, which recognizes specific DNA sequences termed RSS (recombination signal sequences). The RAG recombinase can also catalyze transposition: integration of a DNA segment bounded by RSS into an unrelated DNA target. For reasons that remain poorly understood, such events occur readily in vitro, but are rarely detected in vivo. Previous work showed that non-B DNA structures, particularly hairpins, stimulate transposition. Here we show that the sequence of the four nucleotides at a hairpin tip modulates transposition efficiency over a surprisingly wide (>100-fold) range. Some hairpin targets stimulate extraordinarily efficient transposition (up to 15%); one serves as a potent and specific transposition inhibitor, blocking capture of targets and destabilizing preformed target capture complexes. These findings suggest novel regulatory possibilities and may provide insight into the activities of other transposases.
Highlights
The normal gene-rearranging activities of V(D)J recombination bear a striking resemblance to transposition by certain ‘‘cut-and-paste’’ transposases, including members of the retroviral integrase superfamily [1,2,3] and the hAT family [4]
To determine whether hairpins with other sequences at the tip might act as preferred targets for RAG transposition, we designed a set of 16 selfcomplementary oligonucleotides with all possible fournucleotide combinations surrounding the dyad axis of the inverted repeat
ScaI digestion did yield two distinct products, but only when the cruciforms were extruded (Figure 2D). (Note that even though the ScaI digestion was incomplete, more than 95% of the digestion products appeared in the two expected fragments, indicating that transposition is consistently targeted to the hairpin ends.) Our results clearly show that transposition is targeted to cruciform structures that compete very effectively with the 2.7-kb duplex DNA flanking the cruciforms as transposition targets (Figure 2D)
Summary
The normal gene-rearranging activities of V(D)J recombination bear a striking resemblance to transposition by certain ‘‘cut-and-paste’’ transposases, including members of the retroviral integrase superfamily [1,2,3] and the hAT family [4]. In both reactions, a segment of DNA bounded by specific recognition elements is excised from the chromosome [1,2]. Investigators have proposed a variety of potential regulatory controls that might limit transposition, such as reversal of the reaction by disintegration [7] or inhibition of transposition by the Cterminus of RAG2 [12,13,14] or by GTP [13]
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