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A B-cell-specific DNA recombination complex
T Borggrefe1, M Wabl, A T Akhmedov
1Basel Institute for Immunology, Postfach, CH-4005 Basel, Switzerland.
Insights
Researchers identified the SWAP complex, a multiprotein complex that recombines DNA sequences in immunoglobulin heavy chain switch regions. SWAP-70 protein specifically recruits universal components to form this "switch recombinase".
Area of Science:
- Molecular Biology
- Immunology
- Biochemistry
Background:
- Immunoglobulin (Ig) heavy chain class switching is a critical process in adaptive immunity.
- The molecular machinery responsible for Ig switching, particularly the
- switch recombinase
- , has remained elusive.
Purpose of the Study:
- To purify and biochemically characterize the multiprotein complex involved in Ig heavy chain recombination.
- To identify the specific protein components of this complex and elucidate their roles.
Main Methods:
- Purification and biochemical characterization of the SWAP complex.
- DNA transfer assays to assess recombination activity.
- Protein identification, cDNA cloning, sequencing, and overexpression of SWAP-70.
- Analysis of SWAP-70 expression in B lymphocytes and B-cell lines.
Main Results:
- The SWAP complex preferentially recombines Ig heavy chain switch region sequences.
- Four key proteins identified: B23 (nucleophosmin), C23 (nucleolin), poly(ADP-ribose) polymerase (PARP), and SWAP-70.
- SWAP-70 is a novel 70 kDa nuclear protein expressed specifically in switching B lymphocytes.
- SWAP-70 binds ATP and forms high-affinity complexes with B23, C23, and PARP.
Conclusions:
- The SWAP complex is likely the long-sought
- switch recombinase
- .
- SWAP-70 acts as a specific targeting element, assembling the recombinase from ubiquitous cellular proteins.
- This discovery provides critical insight into the molecular mechanisms of Ig class switching.
Abstract:
We have purified and biochemically characterized a multiprotein complex designated SWAP. In a DNA transfer assay, SWAP preferentially recombines ("swaps") sequences derived from Ig heavy chain switch regions. We identified four of the proteins in the SWAP complex: B23 (nucleophosmin), C23 (nucleolin), poly(ADP-ribose) polymerase (PARP), and SWAP-70. The first three are proteins known to be present in most cells. B23 promotes single-strand DNA reannealing and the formation of joint molecules in a D-loop assay between homologous, but also between Smu and Sgamma sequences. SWAP-70 is a novel protein of 70 kDa. Its cDNA was cloned and sequenced, and the protein was overexpressed in Escherichia coli. SWAP-70 protein expression was found only in B lymphocytes that had been induced to switch to various Ig isotypes and in switching B-cell lines. SWAP-70 is a nuclear protein, has a weak affinity for DNA, binds ATP, and forms specific, high affinity complexes with B23, C23, and poly(ADP-ribose) polymerase. These findings are consistent with SWAP being the long elusive "switch recombinase" and with SWAP-70 being the specific recruiting element that assembles the switch recombinase from universal components.