A3G is recruited to DSB repair foci. (A) H9 cells were irradiated (4 Gy) and probed with anti-A3G and anti-γ-H2AX antibodies at the indicated times. Nuclei were counterstained with DAPI. (B) Irradiated cells were harvested at the indicated times after IR and A3G content in nuclear and cytoplasmic fractions was assessed by Western blotting. α-tubulin and histone H3 were used as cytoplasmic and nuclear markers, respectively. CEM-SS cells not expressing A3G were used as a negative control. Molecular sizes (kDa) are indicated. (C, top): Scheme of the cytidine deamination assay in which C > U deamination in an oligonucleotide forms a restriction enzyme cleavage site after PCR amplification. ND indicates not deaminated; and D, deaminated. (Bottom): Cytidine deaminase activity in extracts of nuclear and cytoplasmic fractions was assessed by incubation with an oligonucleotide substrate containing a single A3G CCC target site (SEco, 80 nt) for 30 minutes at 37°C. DNA sizes (bp) are indicated on the left. PC indicates positive control oligonucleotide; and NC, negative control oligonucleotide.
