Initiation of V(D)J recombination in vivo: role of recombination signal sequences in formation of single and paired double-strand breaks

The EMBO Journal
S B SteenDavid B Roth

Abstract

In V(D)J recombination, double-strand breaks (DSBs) are introduced at recombination signal sequences (RSSs) which consist of three distinct elements: a heptamer, a 12 or 23 nucleotide spacer and a nonamer. Efficient DSB formation requires a 12/23 RSS pair and occurs at both RSS in a temporally coupled fashion (coupled cleavage). It remains unknown which RSS elements are important for coupled cleavage. Furthermore, it has not been established whether some RSS components are critical only for cleavage in cis, with others mainly promoting cleavage in trans at the partner RSS. We investigated these questions by analyzing the effects of RSS mutations on the formation of DSBs in vivo. The abundance of DSBs in cis (at the mutant RSS) and in trans (at the consensus RSS) was determined using an established ligation-mediated PCR assay. We also developed a Southern blotting approach that allows the first direct measurement of dual and single RSS cleavage in vivo. Our results demonstrate that the heptamer, spacer and nonamer elements are all required for coupled cleavage in vivo. These studies also provide evidence for cleavage events involving a single RSS both in mutant substrates and in substrates containing a consensus 12/23 RSS pair.

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Citations

Oct 29, 2003·The Journal of Experimental Medicine·Darryll D DudleyFrederick W Alt
Jul 30, 2002·The EMBO Journal·Jessica M Jones, Martin Gellert
Mar 27, 2001·Nucleic Acids Research·M J Sadofsky
Aug 15, 1997·Genes & Development·E L Beall, D C Rio
Mar 10, 2001·Cold Spring Harbor Symposia on Quantitative Biology·L E HuyeD B Roth
Dec 16, 1998·Immunological Reviews·M S KrangelX P Zhong
Jan 3, 2001·Molecular and Cellular Biology·S B KaleD B Roth
Jun 3, 2000·Annual Review of Immunology·S D FugmannD G Schatz
May 4, 2007·The Journal of Clinical Investigation·Khie KhiongToshio Hirano
Nov 16, 2006·PLoS Biology·Jennifer E PoseyDavid B Roth
Oct 14, 2003·PLoS Biology·Alfred Ian LeeDavid G Schatz
Oct 19, 2001·Proceedings of the National Academy of Sciences of the United States of America·J M Jones, M Gellert
Nov 1, 2003·Proceedings of the National Academy of Sciences of the United States of America·Chun Jeih RyuJianzhu Chen
Oct 10, 2006·Nature Structural & Molecular Biology·Catherine P LuDavid B Roth
Jun 5, 2007·Trends in Immunology·Suzzette M Arnal, David B Roth
Sep 17, 2003·Nature Reviews. Immunology·David B Roth
Jul 10, 2018·Nucleic Acids Research·Nancy Maizels, Luther Davis
Jun 20, 1998·The Journal of Biological Chemistry·S SantagataE Spanopoulou
May 10, 2005·The Journal of Immunology : Official Journal of the American Association of Immunologists·Alexandru OlaruFerenc Livák
Mar 19, 1999·Molecular and Cellular Biology·S B SteenD B Roth
Sep 23, 2003·The Journal of Immunology : Official Journal of the American Association of Immunologists·Alexandru OlaruFerenc Livák
Jun 22, 1999·Molecular and Cellular Biology·T BailinM J Sadofsky
Sep 4, 1998·Cell·D B Roth, N L Craig
May 2, 1998·Science·M MelekD C van Gent

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