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Unusual DNA structures promote genetic instability. One such example is hairpin DNA, which can form from palindromic sequences and triplet repeats, and is also a characteristic intermediate in V(D)J recombination. We previously found that a large 15.3-kb palindrome that was introduced as a transgene into the mouse germline was highly unstable. Although it could be transmitted, the transgene was found to be rearranged in up to 56% of the progeny, and rearrangement events often involved deletion at the center of symmetry. Here, the fine structure of centrally deleted palindromes was sampled by analysis of recombinant junctions isolated from testes DNA, providing further evidence for a model, previously proposed, that accounts for such deletions on the basis of a hairpin-tip nicking activity. In addition to central deletions, gene conversion events were also elevated in the transgenic palindrome. We have now analyzed instability in two mouse sublines in which (as a result of inversion) the transgenic palindrome had been shortened to 4.2 kb. In these sublines, the transgene was still subject to both rearrangement and gene conversion events at a high frequency, similar to the original 15.3-kb palindrome. Recombination was not limited to the sequences constituting the inverted repeat, but was seen to include sequences lying outside the palindrome. As discussed, the salient feature in all of these observations, a high level of genetic change associated with palindromic DNA, underscores the significance of hairpin DNA and hairpin-tip nicking in genome stability.

Open and shut junctions are rare (V(D)J joining products in which site-specific recognition, cleavage and re-ligation of joining signals has been uncoupled from recombination. Here, we investigate the relationship of opening and shutting to recombination in two ways. First, we have tested a series of substrates containing one or two joining signals in an in vivo assay. Opening and shutting can be readily observed in substrates that have only one consensus joining signal. Thus, unlike recombination, the majority of open and shut events do not require interactions between two canonical joining signals. Next we examined two-signal substrates to investigate the effect of signal proximity on the frequency of dual open and shut events. These experiments indicate that at least some of the time opening and shutting can be a two-signal transaction. Together these results point to two mechanistically related, but distinct origins for open and shut joining events. In one case, cutting and closing may occur without interaction between two signals. In the other, we suggest that interaction of a canonical signal with 'cryptic' signal-like elements whose sequence is extensively diverged from canonical signals, may bias the V(D)J recombination machinery towards opening and shutting rather than recombination. Open and shut operations could in this way provide a means whereby mistakes in target recognition by the V(D)J recombination machinery produce a non-recombinant outcome, avoiding deleterious chromosomal rearrangements in lymphoid tissues.