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P nucleotide insertions and the resolution of hairpin DNA structures in mammalian cells

Lewis, Susanna M. (1994) P nucleotide insertions and the resolution of hairpin DNA structures in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America, 91 (4). pp. 1332-1336. ISSN 0027-8424. PMCID PMC43152.

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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.

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Lewis, Susanna M.0000-0002-4183-1906
Additional Information:© 1994 National Academy of Sciences. Communicated by Martin Gellert, November 12, 1993. The author thanks S. Suh for her excellent assistance in the later phases of this work and L. Czyzyk, R. M. L. Yip, and D. Ding for contributions earlier in the study. D. Schatz kindly supplied one of the oligonucleotide primers used in the DNA sequence analysis. H. Lipshitz and J. Campbell provided comments on the manuscript. I would like to thank the above individuals as well as D. Roth and M. Lieber for helpful discussions. This work was funded by American Cancer Society Grant IM-599. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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American Cancer SocietyIM-599
Subject Keywords:V(D)J recombination; end joining; illegitimate recombination; severe combined immunodefIcleucy; scid
Issue or Number:4
PubMed Central ID:PMC43152
Record Number:CaltechAUTHORS:20141208-150820655
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Official Citation:Lewis, S. M. (1994). P nucleotide insertions and the resolution of hairpin DNA structures in mammalian cells. Proceedings of the National Academy of Sciences, 91(4), 1332-1336. doi: 10.1073/pnas.91.4.1332
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:52485
Deposited By: Jason Perez
Deposited On:09 Dec 2014 16:20
Last Modified:04 Jun 2020 19:48

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