RPA Accumulation during Class Switch Recombination Represents 5′–3′ DNA-End Resection during the S–G2/M Phase of the Cell Cycle

SummaryActivation-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.

Graphical AbstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► RPA and Rad51 bind asymmetrically to recombining immunoglobulin genes ► Ig gene DNA breaks are resected as B cells enter the S–G2/M cell-cycle stages ► ATM regulates localized DNA-end resection in G1 ► DNA-end resection in S–G2/M is extensive and ATM independent