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The RAG1-RAG2 recombinase enzyme reconfigures its active site to cleave both DNA strands. This involves unique DNA structures and movements for efficient V(D)J recombination.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Enzyme active sites catalyzing multiple reactions are known.
  • The mechanism of a single nuclease site cleaving both DNA strands of a double helix remained unclear.
  • The RAG1-RAG2 recombinase initiates V(D)J recombination, a critical process in adaptive immunity.

Purpose of the Study:

  • To elucidate the mechanism of site-specific DNA cleavage by the mammalian RAG1-RAG2 recombinase.
  • To understand how a single nuclease active site processes both DNA strands during V(D)J recombination.

Main Methods:

  • Analysis of site-specific DNA cleavage by mammalian RAG1-RAG2 recombinase.
  • Structural and mechanistic investigation of DNA double helix dynamics during enzymatic processing.

Main Results:

  • The RAG1-RAG2 active site reconfigures for two distinct cleavage reactions.
  • The DNA double helix undergoes significant structural changes, including local unwinding and base stacking, not melting, for initial nicking.
  • A global scissor-like motion facilitates the second strand cleavage and hairpin formation.

Conclusions:

  • The RAG1-RAG2 recombinase employs a dynamic mechanism involving active site reconfiguration and distinct DNA structures for sequential DNA strand cleavage.
  • This mechanism allows a single nuclease site to efficiently process the DNA double helix, leading to hairpin formation crucial for V(D)J recombination.