RNase H-sensitive accumulation of APOBEC3B in a nucleolus after DNA damage.

Abstract

Apolipoprotein B mRNA editing catalytic subunit 3B (A3B), a nuclear enzyme that catalyzes cytidine-to-uridine (C-to-U) editing in single-stranded DNA (ssDNA), contributes to genetic diversity in many cancers. A3B is induced or activated by DNA damage owing to a variety of factors; however, the mechanisms by which A3B accesses ssDNA within the genome remain unclear. In this study, we showed that in unstimulated cells, A3B is retained in the nucleoplasm in an RNA-dependent manner. Upon DNA damage induced by camptothecin or actinomycin D (Act D), both targeting topoisomerase I, or by 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), an alkylating agent that generates apurinic/apyrimidinic sites, A3B accumulates at the nucleolar rim and interior. Using confocal microscopy, we assessed the colocalization of A3B with drug-induced R-loops. A3B accumulation was abolished by RNase treatment, implicating R-loops in its localization. However, the S9.6 antibody, commonly used to detect DNA/RNA hybrids, did not identify R-loop-specific signals in the nucleolus, leaving the direct involvement of R-loops in A3B accumulation unresolved. Conversely, immunoprecipitation-mass spectrometry with data-independent acquisition (IP-MS DIA) revealed increased interactions between A3B and RNA helicases such as DDX17 and DDX21, which are known R-loop-binding proteins, following MNNG or Act D treatment. Our results demonstrate that A3B-induced secondary DNA damage occurs in the nucleolus after DNA damage, providing new insights into the acquisition of cancer diversity involving A3B and the DNA damage response in the nucleolus.