Fluorescence resonance energy transfer analysis of recombination signal sequence configuration in the RAG1/2 synaptic complex

Mihai Ciubotaru, Aleksei N. Kriatchko, Patrick Swanson, Frank V. Bright, David G. Schatz

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

A critical step in V(D)J recombination is the synapsis of complementary (12/23) recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins to generate the paired complex (PC). Using a facilitated ligation assay and substrates that vary the helical phasing of the RSSs, we provide evidence that one particular geometric configuration of the RSSs is favored in the PC. To investigate this configuration further, we used fluorescent resonance energy transfer (FRET) to detect the synapsis of fluorescently labeled RSS oligonucleotides. FRET requires an appropriate 12/23 RSS pair, a divalent metal ion, and high-mobility-group protein HMGB1 or HMGB2. Energy transfer between the RSSs was detected with all 12/23 RSS end positions of the fluorescent probes but was not detected when probes were placed on the two ends of the same RSS. Energy transfer was confirmed to originate from the PC by using an in-gel FRET assay. The results argue against a unique planar configuration of the RSSs in the PC and are most easily accommodated by models in which synapsed 12- and 23-RSSs are bent and cross one another, with implications for the organization of the RAG proteins and the DNA substrates at the time of cleavage.

Original languageEnglish
Pages (from-to)4745-4758
Number of pages14
JournalMolecular and Cellular Biology
Volume27
Issue number13
DOIs
StatePublished - Jul 2007

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Fluorescence Resonance Energy Transfer
Protein Sorting Signals
Genetic Recombination
Energy Transfer
Chromosome Pairing
HMGB2 Protein
V(D)J Recombination
High Mobility Group Proteins
HMGB1 Protein
Fluorescent Dyes
Oligonucleotides
Ligation
Proteins
Gels
Metals
Ions

All Science Journal Classification (ASJC) codes

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

Fluorescence resonance energy transfer analysis of recombination signal sequence configuration in the RAG1/2 synaptic complex. / Ciubotaru, Mihai; Kriatchko, Aleksei N.; Swanson, Patrick; Bright, Frank V.; Schatz, David G.

In: Molecular and Cellular Biology, Vol. 27, No. 13, 07.2007, p. 4745-4758.

Research output: Contribution to journalArticle

Ciubotaru, Mihai ; Kriatchko, Aleksei N. ; Swanson, Patrick ; Bright, Frank V. ; Schatz, David G. / Fluorescence resonance energy transfer analysis of recombination signal sequence configuration in the RAG1/2 synaptic complex. In: Molecular and Cellular Biology. 2007 ; Vol. 27, No. 13. pp. 4745-4758.
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