RPA accumulation during class switch recombination represents 5'-3' DNA-end resection during the S-G2/M phase of the cell cycle

Abstract

Activation-induced cytidine deaminase (AID) promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA)-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR), such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG), or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S-G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S-G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S-G2/M phase of the cell cycle.

Figure 1. RPA recruitment to AID, RAG, and I-SceI-induced breaks

(A) RPA occupancy at the Igh locus of 53BP1^−/− (lane I), H2AX^−/− (lane II), and 2AX^−/−UNG^−/−Msh2^−/− (lane III) B cells activated ex-vivo in the presence of LPS+IL-4. Deep-sequencing read densities were normalized to adjust for library size and bin width (reads per million per kb; RPKM). Numbers in parentheses represent average read density in RPKM for the displayed genomic window. (B) TCRα locus showing recruitment of RPA in 53BP1^−/− (lane I) and 53BP1^+/+ (lane III) control thymocytes. Rag2 occupancy is also included in lane II. (C) Kinetics of RPA recruitment at an I-SceI site engineered at Myc intron 1. Activated B cells were transduced with retroviruses expressing ER-I-SceI and cells were harvested at 0, 0.5, 3, or 24h after tamoxifen treatment.

Figure 2. RPA interacts with resected ssDNA downstream of AID or I-PpoI endonuclease

(A) RPA association with + and − DNA strands at the Grap gene locus from IgκAID-53BP1^−/− activated B cells. RPKM values for the specified window (chr11:61,399,129-61,572,079) are provided in parenthesis. (B) RPA bound to Igh locus. As in (A), ChIP-Seq signals were resolved into upper and lower strands. Technical replicates were treated with E. coli ExoI or RecJ exonucleases nuclease prior to deep-sequencing library preparation. (C) PolII binding to + and − strands at the Grap locus. (D) Technical IP replicates from panels A and C samples incubated in the presence of ExoI. (E) Composite diagram showing RPA profiles at 19 I-PpoI mouse genomic sites in 53BP1^−/− MEFs transduced with retroviruses expressing the I-PpoI homing endonuclease.

Figure 3. Rad51 recruitment to AID on- and off-targets

(A) Rad51 accumulation on the upper (+) and lower (−) strand at the Igh locus of IgκAID-53BP1^−/− activated B cells. (B) Rad51 (blue), RPA (red), and translocation profiles at Mir155 and Cd83 genes in chromosomes 16 and 13 respectively from IgκAID-53BP1^−/− B cells. (C) Chromosomal translocations (TSS +/− 2kb) involving Igh or Myc at Rad51-recruiting genes. Chromosomes 12 and 15 carrying the I-SceI sites were excluded from the analysis. Correlation between the two datasets is calculated using Spearman’s ρ. (D) Rad51 levels (TSS +/− 2kb) at RPA+ and RPA− genes in IgκAID-53BP1^−/− B cells.

Figure 4. RPA and γ H2AX recruitment during cell cycle

(A) RPA accumulation in H2AX^−/− activated B cells at the Igh locus during the cell cycle. Samples were stained with Hoechst dye and sorted into G1, S, and G2/M-phased cells. RPKM values for the specified genomic windows are provided in parenthesis. (B) Extent of H2AX phosphorylation (γH2AX) at the Igh locus in wild type (G1, lane I; S, lane II), H2AX^−/− (G1, lane III), or 53BP1^−/− (G1, lane IV; S, lane V) activated B cells. (C) RPA (G1, G2/M) and γH2AX (G2/M) deposition at Igh in cells from H2AX^−/− or 53BP1^−/− respectively. ChIP-Seq data was split into + and − strands.

Figure 5. ATM is required for G1 but not S-G2/M resection

(A) RPA accumulation at Pim1 and IL4Rα loci from 53BP1^−/− activated B cells that were either treated (lower two panels) or not treated (upper panels) with the ATM inhibitor KU-55933. Samples were sorted into G1 or S/G2/M phased cells using the Hoechst dye 33342. Numbers in parenthesis represent RPKM values within the specified genomic windows. (B) RPA accumulation at Igμ and Igγ1 loci in WT, ATMi-, or ATRi-treated B cells. Values represent the RPKM ratio between G1 and S-G2/M-phased cells. (C–D) RPA recruitment to the TCRα in thymocytes (C) or Igh in activated B cells (D) from 53BP1^−/− (upper) or 53BP1^−/−ATM^−/− (lower) mice. (E) RPA and Rad51 recruitment to activated B cells with an intact NHEJ: IgκAID transgenics, AID^+/+, and AID^−/−.

Figure 6. DNA-end resection during CSR

Model showing how AID-mediated breaks at Igh are either processed by C-NHEJ proteins into efficient switch recombination, or are resected by ATM-dependent A-NHEJ in G1 or by the HR in S-G2/M, leading to RPA recruitment and repair. Based on previous work (Shiotani and Zou, 2009), extensive resection in connection with HR is expected to inhibit ATM activity and potentially reduce the formation of chromosomal translocations via A-NHEJ.