Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Activation-induced cytidine deaminase (AID) is crucial for antibody gene diversification through Ig class switch recombination (CSR) and somatic hypermutation (SHM).
• AID can also induce off-target DNA double-strand breaks (DSBs) in non-Ig genes within activated B cells, potentially leading to genomic instability and B cell lymphomas.

Purpose of the Study:

• To identify and characterize off-target DNA sites targeted by AID-induced DSBs.
• To understand the DNA sequence features and repair pathways associated with AID-induced off-target DSBs.

Main Methods:

• Chromatin immunoprecipitation (ChIP) for Nbs1 (a DSB-binding protein) followed by deep sequencing (ChIP-Seq) to map DSBs.
• Analysis of DNA sequence composition at AID-dependent and AID-independent DSB sites.
• Investigation of the role of Msh2 in AID-induced off-target DSBs.

Main Results:

• Hundreds of off-target AID-dependent DSBs were identified.
• Tandem repeats, including long CA repeats (forming Z-DNA) and WGCW hotspots, were significantly enriched at AID-induced DSB sites.
• Msh2 was found to increase the frequency of off-target DSBs, similar to its effect on CSR-related DSBs.
• Off-target DSBs were predominantly two-ended (G1 phase), but a minority were one-ended (S phase), potentially arising from single-strand break repair during replication.

Conclusions:

• Specific DNA sequence motifs, such as Z-DNA forming repeats and AID hotspots, are preferential targets for AID-induced off-target DSBs.
• The mismatch repair protein Msh2 plays a role in promoting AID-induced off-target DSBs.
• One-ended off-target DSBs generated during replication can be repaired by homologous recombination pathways, potentially leading to genome instability and contributing to B cell lymphomagenesis.