tion of Human Subjects is FWA00003327. The anonymous healthy donors provided their written informed consent to participate in this study. Peripheral blood mononuclear cells were isolated by order Amezinium metilsulfate Ficoll gradient. Isolation of CD4+ T lymphocytes was performed by incubation with antibody-coated magnetic beads. Purity of CD4+ T lymphocytes was above 90% as checked by flow cytometry. CD4+ T lymphocytes were stimulated with 10 g ml-1 PHA, 100 U ml-1 IL2 and 1000 U ml-1 IFN for 48 h. Materials and Methods A3A isoforms p1 and p2 The cDNAs encoding the two A3A isoforms were those corresponding to the sequence Genbank Accession Number NM_145699. Primers were designed to equip both A3A isoforms with adequate and strong Kozak motifs respectively. For one construction the SV40 TAg nuclear localization signal was added to the C-terminus. Full-length cDNAs were subcloned in the pcDNA3.1D/V5-HisTOPO expression vector. All constructs were transformed and amplified in E. coli DH5 strain. 
Catalytically inactive A3A mutants were made by engineering C101S or C106S substitutions into active site residues, following manufacturer’s recommendation. PCR and 3DPCR All DNAs were extracted using the MasterPure Complete DNA and RNA purification kit. Amplifications were performed in a first-round standard PCR followed by nested 3DPCR with 2.5 U Taq DNA polymerase per reaction. PCR products were cloned using the TOPO TA Cloning kit while sequencing was outsourced to GATC. PCR conditions and primers have been described. For the detection of hypermutation by 3DPCR, primary cells were infected with lentivirus rV2.EF1.UGI, which encodes a codon optimized UNG inhibitor under the control of the constitutive human EF1 promoter generated by Vectalys. Stock virus was pseudotyped with the VSV G protein. Purified human CD4+ T lymphocytes were transduced by polybrene at the MOI of five according to the manufacturer’s instruction. 2 APOBEC3A Isoforms Induce DNA Damage and Apoptosis Flow cytometry of DNA damage response Twenty-four and 48 h post transfection floating and adherent cells were washed with PBS, fixed in 2% 12414725 ice-cold paraformaldehyde for 15 min and permeabilized in 90% ice-cold methanol for 30 min. After two washes with PBS, cells were incubated for 1 h with 1:200 diluted mouse anti-V5 antibody. DNA double strand breaks were analysed by staining for 1 h with 1:50 diluted Alexa Fluor 488-conjugated rabbit monoclonal antiH2AX antibody. Phosphorylated Chk2 was detected by using 1:50 diluted rabbit monoclonal antiChk2-P antibody for 1 h. Detection of cleaved PARP was performed by incubation with 1:50 diluted Alexa Fluor 488-conjugated rabbit monoclonal anti-cleaved PARP antibody. Following secondary antibodies were: 1:500 diluted Alexa Fluor 633 F2 fragment of goat anti-mouse IgG , 1:100 diluted FITC goat anti-mouse IgG or 1:100 diluted FITC goat antirabbit IgG for 45 min. All incubation steps were performed on ice. Cells were analysed on FACSCalibur using CellQuest Pro or FlowJo software. For each sample 10,000 cells were counted. were collected. Cellular aggregates and debris were excluded from analysis by proper gating. Data were fit to define the G1, S, and G2/M phases by using the Dean-Jett-Fox mathematical model of the FlowJo software. Mitochondrial cytochrome c release At 24 h post transfection, HeLa cells were trypsinized and investigated for cytochrome 14707029 c release by using the FlowCellect cytochrome c kit from Millipore according to manufacturer’s instructions. Cells were
