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Ordered assembly of the V(D)J synaptic complex ensures accurate recombination
Jessica M Jones1, Martin Gellert
1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5/Room 241, Bethesda, MD 20892, USA.
The EMBO Journal
|July 30, 2002
Summary
The RAG1/2 complex initiates DNA cleavage by assembling on one DNA signal sequence (RSS) first. This initial assembly dictates the subsequent binding of the complementary RSS, ensuring precise gene recombination.
Area of Science:
- Molecular Biology
- Immunology
- Genetics
Background:
- V(D)J recombination is crucial for adaptive immunity, enabling the generation of diverse immunoglobulin and T-cell receptor genes.
- The recombination-activating gene (RAG) proteins, RAG1 and RAG2, are essential for initiating this process by binding and cleaving specific DNA signal sequences (RSSs).
- RSSs contain conserved nonamer and heptamer sequences flanking a spacer of either 12 or 23 base pairs, and their precise joining is critical for functional gene rearrangement.
Purpose of the Study:
- To investigate the assembly mechanism of the RAG1/2 synaptic complex and its implications for DNA cleavage activity.
- To determine the order of RSS binding and its effect on the efficiency and specificity of V(D)J recombination.
- To elucidate the role of the 12/23 spacer rule in guiding the RAG1/2 complex assembly and function.
Main Methods:
- In vitro assembly of RAG1/2 multimers on isolated DNA signal sequences (RSSs) with 12- and 23-bp spacers.
- Analysis of synaptic complex formation using purified RAG1/2 proteins and RSS substrates.
- Assessment of DNA cleavage activity in response to different orders of RSS binding.
Main Results:
- A RAG1/2 multimer capable of cleaving both 12 and 23 RSSs assembles on an isolated RSS, with the complementary RSS entering as naked DNA.
- Pre-binding of RAG1/2 to both 12 and 23 RSSs separately significantly reduces synaptic complex assembly and cleavage activity.
- RAG1/2 complexes assembled on a 12 RSS show a preference for incorporating a 23 RSS partner, while complexes on a 23 RSS prefer a 12 RSS partner, supporting the 12/23 rule.
Conclusions:
- The initial assembly of the RAG1/2 complex on a single RSS is essential for productive DNA cleavage and V(D)J recombination.
- The observed binding preferences of RAG1/2 complexes for complementary RSS partners reinforce the strict 12/23 spacer joining rule.
- Cellular factors, potentially including chromatin, may guide the RAG1/2 complex to initiate assembly on a 12 RSS, followed by the recruitment of a 23 RSS to activate cleavage.