Lewis S M
Division of Biology, California Institute of Technology, Pasadena 91125.
Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1332-6. doi: 10.1073/pnas.91.4.1332.
Two lines of evidence point to a hairpin DNA intermediate in V(D)J joining (V, variable; D, diversity; J, joining) [Lieber, M.R. (1991) FASEB J. 4, 2934-2944]. One is the presence of P nucleotide insertions (short inverted-repeat sequence) in V(D)J junctions [Lafaille, J. J., DeCloux, A., Bonneville, M., Takagaki, Y. & Tonegawa, S. (1989) Cell 59, 859-870]; a second is the detection of site-specifically broken DNA molecules with covalently closed (hairpin) termini in thymus DNA [Roth, D. B., Menetski, J. P., Nakajima, P., Bosma, M. J. & Gellert, M. (1989) Cell 70, 983-991]. However, P nucleotide insertions could be generated in ways not involving a hairpin structure, and because physical evidence for hairpin-ended DNA fragments has been obtained only with mutant mice, there is some uncertainty regarding the role of hairpin molecules in the normal V(D)J joining pathway. To determine whether mammalian cells are capable of metabolizing this odd type of DNA terminus and whether, in doing so, junctions with P insertions are in fact created, a linear DNA molecule with a hairpin closure at each end was transfected into several murine cell lines. The hairpin-ended molecules were recircularized, and the junctions exhibited P insertions at a high frequency. This result directly links the presence of P insertions to a hairpin precursor, providing strong evidence for the notion that a hairpin DNA intermediate exists in V(D)J recombination. A comparison of hairpin end joining in various cells, including those derived from mice with the severe combined immunodeficiency (scid) mutation, is presented.
有两条证据指向V(D)J连接（V，可变区；D，多样区；J，连接区）过程中存在发夹状DNA中间体[利伯，M.R.（1991年）《美国实验生物学会联合会杂志》4，2934 - 2944]。一条是V(D)J连接点处存在P核苷酸插入（短反向重复序列）[拉法伊尔，J.J.，德克洛，A.，博纳维尔，M.，高垣，Y. & 利根川进（1989年）《细胞》59，859 - 870]；另一条是在胸腺DNA中检测到具有共价封闭（发夹）末端的位点特异性断裂DNA分子[罗斯，D.B.，梅内茨基，J.P.，中岛，P.，博斯马，M.J. & 盖勒特，M.（1989年）《细胞》70，983 - 991]。然而，P核苷酸插入可能通过不涉及发夹结构的方式产生，并且由于仅在突变小鼠中获得了发夹末端DNA片段的物理证据，关于发夹分子在正常V(D)J连接途径中的作用存在一些不确定性。为了确定哺乳动物细胞是否能够代谢这种奇特类型的DNA末端，以及在这样做的过程中是否实际上产生了带有P插入的连接点，将两端带有发夹封闭结构的线性DNA分子转染到几种小鼠细胞系中。带有发夹末端的分子重新环化，并且连接点以高频率出现P插入。这一结果直接将P插入的存在与发夹前体联系起来，为V(D)J重组中存在发夹状DNA中间体这一观点提供了有力证据。本文还比较了各种细胞中的发夹末端连接情况，包括来自具有严重联合免疫缺陷（scid）突变小鼠的细胞。
