Quantitative analyses of RAG-RSS interactions and conformations revealed by atomic force microscopy

Area of Science:

• Molecular Biology
• Genetics
• Biophysics

Background:

• V(D)J recombination is crucial for adaptive immunity.
• RAG1/2 proteins bind recombination signal sequences (RSS) to initiate DNA cleavage.
• DNA bending is hypothesized to facilitate RAG-RSS interaction and cleavage.

Purpose of the Study:

• To quantitatively measure DNA bending induced by RAG1/2 proteins at RSS using atomic force microscopy (AFM).
• To investigate the structural role of RAG2 in RAG-RSS complex formation and DNA bending.
• To assess DNA conformation changes after nicking of the RSS.

Main Methods:

• Atomic Force Microscopy (AFM) imaging of individual RAG-RSS complexes.
• Quantitative analysis of DNA bending angles in RAG-12RSS and RAG1/2-12RSS complexes.
• Comparison of DNA conformation between intact and prenicked 12RSS bound by RAG1/2.

Main Results:

• RAG1 alone was sufficient to induce significant DNA bending in the 12RSS.
• No significant difference in DNA bending was observed between RAG1-12RSS and RAG1/2-12RSS complexes.
• A prenicked 12RSS bound by RAG1/2 showed a similar conformation to intact 12RSS, indicating no increased bending after nicking.
• The RAG-RSS complex exhibits a stable bend angle of approximately 60 degrees.

Conclusions:

• RAG1 is the primary driver of DNA bending in the RAG-RSS complex.
• RAG2 does not appear to significantly alter the DNA bending conformation.
• The ~60-degree DNA bend angle is a critical prerequisite for V(D)J recombinase-mediated DNA nicking.