R-loop disruption triggers irreversible topoisomerase 1-DNA cleavage complex formation during AID-dependent IgH gene...

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Antibody production relies on class switch recombination (CSR) and somatic hypermutation, mediated by activation-induced cytidine deaminase (AID).
• Topoisomerase 1 (TOP1) is hypothesized to facilitate AID-dependent DNA cleavage, but the mechanism of TOP1-DNA cleavage complex (TOP1-CC) formation after AID's C-to-U editing is unclear.

Purpose of the Study:

• To elucidate the molecular mechanism linking AID-mediated C-to-U editing to TOP1-CC formation during CSR.
• To investigate the role of R-loops and their destabilization in this process.

Main Methods:

• Utilized antisense oligonucleotides (ASOs) targeting germline transcripts (GLTs) to disrupt R-loops at DNA cleavage sites during CSR.
• Performed in vitro transcription assays with RNase H to assess R-loop degradation effects on supercoiling and double-strand breaks.
• Generated and analyzed Tyrosyl-DNA phosphodiesterase 1 (TDP1) knockout cells to study TOP1-CC processing and its impact on CSR.

Main Results:

• ASO-mediated R-loop disruption increased DNA cleavage frequency and TOP1-CC formation within GLT regions during CSR.
• RNase H degradation of R-loops enhanced TOP1-mediated supercoiling and double-strand breaks independently of AID.
• TDP1 deficiency suppressed CSR, suggesting accumulated TOP1-CCs are processed via an alternative pathway.

Conclusions:

• R-loop destabilization is a critical, previously unrecognized intermediate step connecting AID's C-to-U editing to TOP1-CC formation.
• AID editing within R-loops creates G:U wobble base pairs, destabilizing R-loops and leading to their disruption by nuclear RNases.
• This R-loop disruption induces TOP1-CCs, facilitating CSR through a mechanism involving alternative TOP1-CC processing pathways.