DNA melting initiates the RAG catalytic pathway

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The initiation mechanism of DNA cleavage by DDE-family enzymes, crucial for V(D)J recombination, remains unclear.
• The RAG endonuclease plays a key role in V(D)J recombination, a process essential for adaptive immunity.

Purpose of the Study:

• To elucidate the mechanism of DNA cleavage initiation by the RAG endonuclease.
• To understand the structural basis of RAG's interaction with recombination signal sequences (RSSs).

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structures of zebrafish RAG.
• Complexes of RAG with one or two intact RSSs were analyzed at high resolution (up to 3.9 Å).

Main Results:

• Cryo-EM structures revealed unexpected DNA melting at the RSS heptamer, leading to DNA rotation and positioning of the scissile phosphate in the active site.
• RAG1 undergoes significant conformational changes, including piston-like movements, upon substrate binding to accommodate and wedge melted DNA.
• Limited base-specific contacts were observed, suggesting DNA unwinding propensity is key for conserved sequences.

Conclusions:

• The study proposes a universal DNA melting mechanism initiated by RAG, conserved in V(D)J recombination, DNA transposition, and retroviral integration.
• The findings provide critical insights into the structural dynamics of RAG-RSS complexes and the initiation of DNA cleavage.