P-NUCLEOTIDE INSERTIONS AND THE RESOLUTION OF HAIRPIN DNA STRUCTURES IN MAMMALIAN-CELLS

Two lines of evidence point to a hairpin DNA intermediate in V(D)J joi ning (V, variable; D, diversity; J, joining) [Lieber, M. R. (1991) FAS EB J. 4, 2934-2944]. One is the presence of P nucleotide insertions (s hort inverted-repeat sequence) in V(D)J junctions [Lafaille, J. J., De Cloux, A., Bonneville, M., Takagaki, Y. & Tonegawa, S. (1989) Cell 59, 859-870]; a second is the detection of site-specifically broken DNA m olecules with covalently closed (hairpin) termini in thymus DNA [Roth, D. B., Menetski, J. P., Nakajima, P., Bosma, M. J. and Gellert, M. (1 989) Cell 70, 983-991]. However, P nucleotide insertions could be gene rated in ways not involving a hairpin structure, and because physical evidence for hairpin-ended DNA fragments has been obtained only with m utant mice, there is some uncertainty regarding the role of hairpin mo lecules in the normal V(D)J joining pathway. To determine whether mamm alian cells are capable of metabolizing this odd type of DNA terminus and whether, in doing so, junctions with P insertions are in fact crea ted, a linear DNA molecule with a hairpin closure at each end was tran sfected into several murine cell lines. The hairpin-ended molecules we re recircularized, and the junctions exhibited P insertions at a high frequency. This result directly licks the presence of P insertions to a hairpin precursor, providing strong evidence for the notion that a h airpin DNA intermediate exists in V(D)J recombination. A comparison of hairpin end joining in various cells, including those derived from mi ce with the severe combined immunodeficiency (scid) mutation, is prese nted.