Somatic hypermutation of human mitochondrial and nuclear DNA by APOBEC3 cytidine deaminases, a pathway for DNA catabolism

Rodolphe Suspènea,1, Marie-Ming Aynauda,1, Denise Guétarda, Michel Henrya, Grace Eckhoffa, Agnès Marchiob, Pascal Pineaub, Anne Dejeanb,c, Jean-Pierre Vartaniana, and Simon Wain-Hobsona,2

aInstitut Pasteur, Molecular Retrovirology Unit, Centre National de la Recherche Scientifique URA3015 and bNuclear Organization and Oncogenesis Unit, 75724 Paris cedex 15, France; and cInstitut National de la Santé et de la Recherche Médicale, U579, 75724 Paris cedex 15, France

Edited by Reuben S. Harris, University of Minnesota, Minneapolis, MN, and accepted by the Editorial Board February 3, 2011 (received for review July 7, 2010) The human APOBEC3 (A3A–A3H) locus encodes six cytidine deam- As there are six human A3 enzymes, all with ssDNA specificity, inases that edit single-stranded DNA, the result being DNA pep- the question arises as to whether they can edit host-cell DNA. pered with uridine. Although several cytidine deaminases are Given the high mtDNA copy number per cell, we explored the clearly restriction factors for retroviruses and hepadnaviruses, it possibility that a fraction of mtDNA might be vulnerable to A3 is not known if APOBEC3 enzymes have roles outside of these set- editing, especially in inflamed tissues where there is demonstrable tings. It is shown here that both human mitochondrial and nuclear up-regulation of A3 genes (17). We show here that a small frac- DNA are vulnerable to somatic hypermutation by A3 deaminases, tion of human mitochondrial genomes are phenomenally edited with APOBEC3A standing out among them. The degree of editing is by APOBEC3 deaminases in the cytoplasm. It transpires that much greater in patients lacking the uracil DNA-glycolyase gene, nuclear DNA (nuDNA), notably segments of MYC and TP53, can indicating that the observed levels of editing reflect a dynamic be hyperedited by APOBEC3A. For both mitochondrial and composed of A3 editing and DNA catabolism involving uracil nuDNA, editing reflects a dynamic between A3 activity and DNA-glycolyase. Nonetheless, hyper- and lightly mutated sequen- DNA degradation involving UNG.

ces went hand in hand, raising the hypothesis that recurrent low- CELL BIOLOGY level mutation by APOBEC3A could catalyze the transition from Results a healthy to a cancer genome. APOBEC3 Hypermutation of mtDNA. A nested PCR/3DPCR ap- proach was taken to the amplification of mtDNA from 36 DNA he human APOBEC3 (A3) locus encodes seven expressed samples spanning alcoholic, HBV+, HBV+HCV+, and HCV+ Tgenes (A3A–A3H), of which six are demonstrably cytidine cirrhosis (17, 21). A region corresponding to part of MT-COI deaminases with specificity for single-stranded DNA (ssDNA) at ∼6,200 bp was chosen, as it maps within the large single- (1). The A3 locus arose in placental mammals by duplication of stranded replication intermediate. Mitochondrial DNA could be AICDA and subsequent expansion (2). Activation-induced cyti- recovered at temperatures as low as 82.2 °C from 17 of 36 (∼47%) dine deaminase (AICDA) is presently the only enzyme known to samples tested, much lower than for a control liver or cloned MT- induce mutations in the human genome and initiates class-switch COI DNA (Td∼86 °C) (Fig. 1 A and B). Molecular cloning and recombination and somatic hypermutation of rearranged Ig genes sequencing of the PCR products proved that they represented bona (3). Several human A3 genes are up-regulated in inflammatory fide cytidine-edited mtDNA, with both strands being vulnerable settings by type I and II interferons, although activation depends (Fig. 1C and Fig. S1A), although minus-strand hypermutants to some degree on the cell type (4–8). Given this finding, it is not were generally more abundant (shown as G->A hypermutants with surprising that A3F and A3G are involved in the restriction of respect to the plus strand). Although minus-strand editing was retroviruses and hepadnaviruses where nascent single-stranded generally in the 30 to 80% range, occasionally a sequence with up to cDNA remains vulnerable until double-strand synthesis is com- 98% of targets could be edited (Fig. 1 C and D). – pleted (9 16). In late stages of hepatitis B virus- (HBV) associ- As A3 and AICDA catalyzed cytidine deamination is partic- fi fi ated cirrhosis there was signi cant up-regulation of four to ve A3 ularly sensitive to the 5′ base, a bulk dinucleotide analysis was genes, with A3G being the major restriction factor able to edit performed for minus- and plus-strand hypermutants (Fig. 1 E up to 35% of HBV genomes (17). and F). The strong biases for TpC and CpC among minus-strand DNA viral genomes, such as human papillomavirus (HPV), hypermutants are hallmarks of APOBEC3 editing (11, 15, 22). can also undergo editing both in vivo and in tissue culture, For plus-strand editing, the clear preference for CpC is a hall- presumably during transcription or replication (18). Three A3 mark of A3G (Fig. 1F), no matter the template (11). These deaminases, A3A, A3C, and A3H could hyperedit HPV genomes slightly different patterns probably reflect the greater C content ex vivo, suggesting that the phenomenon was occurring in the of the plus strand generating more oligoC tracts, which are nucleus because the strictly cytoplasmic deaminases, A3F and known hotspots for A3G. A clonal analysis of the dinucleotide A3G, did not edit transfected HPV DNA (18). Along with a re- cent article showing that A3A could edit transfected EGFP plasmid DNA (7), they highlight the potential of A3 enzymes to Author contributions: R.S., J.-P.V., and S.W.-H. designed research; R.S., M.-M.A., D.G., edit foreign DNA. The presence of uridine in cellular DNA is M.H., G.E., and A.M. performed research; P.P. and A.D. contributed new reagents/analytic read as a danger signal, the uracil base resulting from cytidine tools; R.S., M.-M.A., J.-P.V., and S.W.-H. analyzed data; and J.-P.V. and S.W.-H. wrote the deamination being subject to excision, particularly by uracil paper. DNA-glycosylase (UNG), the nuclear form (UNG2) being far The authors declare no conflict of interest. more active than its mitochondrial counterpart (UNG1). Uracil This article is a PNAS Direct Submission. R.S.H. is a guest editor invited by the Editorial excision followed by abasic endonuclease cleavage of the DNA Board. strand can lead to repair or degradation. Small fragments of nu- 1R.S. and M.-M.A. contributed equally to this work. clear and mitochondrial DNA (mtDNA) can be found in the 2To whom correspondence should be addressed. E-mail: [email protected]. cytoplasm, which trigger a number of cytoplasmic DNA sensor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. molecules and are associated with DNA degradation (19, 20). 1073/pnas.1009687108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1009687108 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 Fig. 1. Somatic hypermutation of mitochondrial DNA in cirrhosis. (A) Agarose gels of COI 3DPCR products for DNA from one control and two virally infected livers with cirrhosis. M, molecular weight markers. (B) Schematic representing the denaturation temperature for the last positive 3DPCR amplifications for a variety of liver samples. COI DNA indicates a molecular clone of the reference sequence. (C) A selection of hypermutated COI sequences from cirrhosis sample #166, Td = 84.6 °C. Although editing occurs on both strands, the sequences are given with respect to the plus or coding strand. (D) Distribution of minus-strand hyperedited mtDNA sequences (Td = 84.6 °C) derived from 10 different cirrhosis samples. (E) Bulk 5′ dinucleotide analysis for the G->A hypermutants from three patients. Letters indicate statistical significance in a χ2 test. (F) Bulk 5′ analyses for plus-strand hypermutants (C->T). (G)A5′ dinucleotide analysis of individual sequences (clonal analysis) showing that all mapped to the region typical of A3 deaminases. The number of TpC+CpCvs. GpC+ApC targets edited per sequence are computed and represented on the y and x axes, respectively. Color coding is the same as in E.

context for three liver samples showed that all edited mtDNA neutrophils showing the greatest degree of editing: that is, sequences mapped in the region typical of A3 deaminases (Fig. 3DPCR products were recovered as low as 82.2 °C (Fig. 2B). 1G), meaning that there was not also a handful of AICDA-edi- Sequencing showed that cytidine deamination was concentrated ted sequences, which would show up in the GpC+ApC region, in TpC and CpC motifs for all cell subsets, indicating that the hidden among a majority of A3-edited sequences. same A3, or ensemble of A3 enzymes, was responsible (Fig. 2D). Numerous human cell lines are known to express A3 genes (1) Mitochondrial DNA Editing in Peripheral-Blood Mononuclear Cells. and eight of eight cell lines tested positive for hyperedited Virus-infected tissues can be infiltrated by leukocytes, but nu- mtDNA (Fig. 2C). To get an independent idea of the frequencies merous studies have described A3 expression in resting and acti- of mtDNA editing in PBMCs, we sequenced ∼1,000 clones vated leukocytes (5, 6, 23). To explore this issue, total DNA from from standard PCR for two samples (AS0488 and AS2017) 11 Ficoll-purified peripheral-blood mononuclear cells (PBMCs) (Table S1) yet did not find a single hypermutated sequence in- − and 3 Epstein Barr virus (EBV)-transformed PBMCs was ana- dicating hypermutant frequencies of <10 3. lyzed (Table S1). In all cases, 3DPCR-recovered edited mtDNA way below the reference temperature of ∼86 °C (Fig. 2A). This Mitochondrial DNA Editing Occurs Outside of the Mitochondrion. The finding is interesting in that the samples spanned a broad age intracellular localization of transfected A3-V5 tagged constructs range: from a 3-wk-old child without pathology to nonagenerians was analyzed by confocal microscopy using Mitrotracker deep (Table S1). Sequence analysis confirmed A3 hyperediting (i.e., red. Despite extensive screening, we were unable to find any there was a strong bias in favor of TpC and CpC, typical of A3 colocalization of any A3 with the mitochondrial network, which deaminases). Citrate-treated blood from two donors was Ficoll- would show up as strong diagonals (Fig. 2E), suggesting that fractionated using antibody-coated magnetic beads to purify editing occurred in the cytoplasm. To explore this theory, HeLa CD4+ and CD8+ T cells, CD14+ monocytes, CD15+ neutrophils, cells were treated with 0.5% Triton X-100 and incubated with 1 CD16+ eosinophils+basophils, CD19+ B cells, and CD56+ nat- μg/mL of DNaseI for 1 h, which removed the hyperedited mtDNA ural killer cells. MT-COI editing was evident for all fractions, the yet left a strong normal mtDNA signal (Fig. 2F). As DNase

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1009687108 Suspène et al. Downloaded by guest on October 1, 2021 CELL BIOLOGY

Fig. 2. Mitochondrial DNA editing occurs among PBMCs and in the cytoplasm. (A) Schematic representation for 3DPCR amplification for human PBMCs, EBV- transformed PBMCs, and purified sperm. (B) Comparable representation for PBMC subsets from donors ABD2 and -4. (C) Comparable representation for established human cell lines. The A3C1 and WTI lines are derivatives of Jurkat. (D) Clonal analysis of edited mtDNA sequences for PBMC cell subsets from donor ABD4. Color coding corresponds to that in B.(E) Confocal microscopic analysis of double positive A3-V5 transfected HeLa cells plus Mitotracker deep red. The absence of a diagonal in any plot indicates a lack of colocalization. (F) 3DPCR temperature-gradient agarose gels revealing recovery of edited mtDNA. The legend to the right describes the treatment, transfection, and sample; ± DNaseI-treated HeLa cells lyzed by Triton X-100 indicating that editing occurs in the cytoplasm; Taq/Taq or Pfu/Taq indicates first-round amplification using Taq or Pfu DNA polymerase followed by Taq for 3DPCR. Pfu is unable to copy DNA containing dU; pv, empty plasmid; UGI refers to the phage UNG inhibitor; P1-3 are human ung−/− cell lines and P1 PBMCs indicates total PBMC DNA from patient P1. (G) Schematic of last positive 3DPCR amplification temperature for the quail QT6 cell line transfected by a series of APOBEC plasmids in duplicate. The prefixes “h” and “m” denote human and mouse, respectively. Both hA3Gn and hA3Gc denote the N and C domains alone of hA3G; the C288S and C291S mutants of hA3G are inactive.

treatment alone did not reduce the edited DNA signal, this Given this finding, we next turned to EBV transformed B-cell − − rules out extracellular mtDNA as a source for the signal (Fig. 2F). lines from three ung / individuals [P1, P2, and P3 (26)]. If hyperediting occurred outside of the mitochondrion, it should Hyperedited mtDNA was detected for all three samples down to not be subject to DNA repair. Like all archaeabacterial poly- 81 °C, confirming the results had with UGI (Fig. 2F). To de- merases, Pfu is unable to amplify DNA harboring dU (24). We termine A3 expression levels, real-time quantitative PCR was fi fi performed 3DPCR on rst round PCR mtDNA ampli ed at 95 °C performed for P1, P2, and P3, as well as two ung+ control EBV- − − using Pfu. This strategy failed to recover any genomes beyond transformed cell lines, Z (AICDA / ) and T (Fig. S2A). Data the 85.4 to 86 °C benchmark temperature for unedited mtDNA were normalized to the expression levels of four invariable ref- (Fig. 2F). These findings show that A3 editing of mtDNA occurs erence genes (CLK2, GUSB, HMBS, TBP). Although A3 ex- outside of the mitochondrion and goes uncorrected. pression was in general similar, A3A showed some variation The high levels of hyperedited mtDNA in established cell lines has thwarted detailed analysis of the phenomenon by trans- among the samples (Fig. S2A). Analysis of total PBMC DNA fection experiments. Only for A3A transfections, and to a lesser from one of these individuals (P1) yielded the same result as the fl extent A3G, could a clear signal over background be found (Fig. cell line, ruling out any in uence from EBV on the observation. 2F). Uracil-DNA glycosylase inhibitor (UGI) is a small protein For P2 DNA, the frequency of mtDNA hypermutants in stan- from a Bacillus subtilis bacteriophage that can inhibit mammalian dard PCR products was assessed by deep sequencing. Approxi- UNG, a crucial enzyme involved in excising uracil from DNA mately 0.5% were edited, greater than for PBMC DNA from (25). When A3A or A3G were cotransfected with the UGI plas- normal individuals (vide supra). This finding translates to ∼25 per mid, mtDNA recovered at temperatures as low as 81 °C (Fig. 2F). cell, assuming ∼5 K mtDNA genomes per cell.

Suspène et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 Limiting-dilution of PBMCs and purified CD4 cells from pa- other mammals, the A3 gene number presently ranges from one tient ABD2 were performed in triplicate, the hyperedited (mice, rats, pigs), to three (cats) and six (horses) (27). In con- − 3DPCR signal being lost between 1,000 and 300 cells (f ≥ 10 3). trast, birds, reptiles, and fish do not encode an A3 gene. Edited A similar triplicate titration of P2 cells resulted in a loss of signal mtDNA was recovered from tree shrew hepatocyte cultures − between 50 and 10 cells (f ≥ 2 × 10 2). Given 3DPCR as readout, (Tupaia belangeri belongs to Scandentia, the closest order to these frequencies are underestimates. For P2 cells where the Primates) as well as horse and goat PBMCs (Table S2). In all frequencies of cells harboring edited mtDNA and bulk hyper- cases, 5′ dinucleotide analysis revealed a penchant for editing edited mtDNA are known, it can be concluded that a sizeable within TpC and CpC, typical of A3 enzymes. Mouse DNA from fraction of cells harbors a small fraction of hyperedited mtDNA numerous tissues was systematically negative for mtDNA editing sequences. (Table S2). These findings show that the phenomenon of All of the above analyses concerned editing of the the MT-COI mtDNA editing is not uniform across placental mammals. gene. To be sure that there was no bias, part of the MT-CYB and MT-ND2 genes from the cell lines P1, P2, and P3 were analyzed A3 Editing of the Human nuDNA. As the A3A, A3C, and A3H by 3DPCR. Like MT-COI, the former maps to the ssDNA rep- enzymes in particular can locate to the nucleus, we tested the lication intermediate but MT-ND2 mapped to the dsDNA re- possibility that, occasionally, nuDNA might be vulnerable to A3 gion. Both DNA strands of MT-CYB and MT-ND2 were heavily editing. MYC is particularly mutated in leukemias and lympho- edited; a selection from P2 cells is shown in Fig. S1B. mas, and the tumor-suppressor gene TP53 is mutated in ∼50% of Although A3A and A3G can edit mtDNA, this doesn’t exclude cancers, with exon 8 encoding many hotspots (http://p53.free.fr). other A3 enzymes that could be individually, or together, re- Several of the human cirrhosis and PBMC samples where sponsible for the high background. To address this question we mtDNA editing was in evidence were screened using primers for used the QT6 quail cell line that has never shown an endogenous MYC exon 2 and TP53 exon 8/intron 9. We were unable to re- cytidine-editing background for retroviruses, presumably because cover massively hypermutated MYC or TP53 sequences. the avian lineage does not encode A1 or A3 othologs (11). QT6 Given the impact of UNG on mtDNA editing (Fig. 2F), we − − cells were transfected by numerous APOBEC plasmids. The turned to the three human ung / cell lines [P1, P2, and P3 (26, PCR/3DPCR approach recovered hyperedited quail mtDNA for 28)]. Hyperedited MYC DNA was recovered as low as 91 °C from 5 of 11 human cytidine deaminases, notably A3A, A3C, A3F, all three cell lines, as well as from P1 PBMC DNA (Fig. 3A). A3G, and A3H; the murine A1 and A3 enzymes were also capable Sequencing revealed a range of editing, from 1 to 38 C->T of editing QT6 mtDNA (Fig. 2G). transitions per strand (Fig. 3B), with a strong penchant for TpC Given this result, it is possible that mtDNA editing may occur and CpC (Fig. 3 C–E). The frequency of hyperdited MYC beyond the primate world, with its seven-gene A3 locus. For sequences can be estimated from the number of unique sequences

Fig. 3. Somatic hypermutation of MYC DNA. (A) Agarose gel of 3DPCR products from the P1-3 cell lines and total DNA from P1-derived PBMCs. The 3DPCR products recovered at temperatures lower than the white vertical line are edited. (B) A selection of hypermutated MYC exon-2 sequences; only differences are shown with respect to the reference sequence. For clarity, only 170 of the 241-bp sequences are shown and the numbers and percent of mutations refers to the complete sequence. (C and D)Bulk5′ dinucleotide analysis of editing for the C->TandG->A hypermutants respectively. χ2 significance is as in Fig. 1E.(E) Clonal analysis of C->T(red)andG->A (blue) hypermutants. The red and blue diagonals represent the expected values based on the target dinucleotide composition.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1009687108 Suspène et al. Downloaded by guest on October 1, 2021 derived from 0.5-μg input DNA (∼150 K copies). As ∼30 to 50% teaued at ∼20% of cytidines deaminated at ∼2 μM of enzyme (32), of sequences were identical, the number of distinct sequences is in meaning that the hyperediting observed cannot be simply the the range of the number of unique sequences. Hence, the chance encounter of free enzyme and target ssDNA in a per- − hyperedited MYC frequencies are in the range of 20/150 K ∼10 4. meablized or collapsed cell. The Triton-dependent DNase sensi- At such low frequencies, correlations with macroscopic, such as tivity of edited mtDNA supports this deduction. Hypermutated bulk transciption, levels are going to be fraught with problems. human DNA therefore implies compartmentalization or cofactors To explore nuDNA editing experimentally, we transfected where ssDNA and A3 remain together, rather than permitted to HeLa cells with individual A3-V5 constructs ±UGI and sought drift apart, and so contributing to the efficiency of the catabolic hyperedited MYC DNA at 72 h posttransfection. Only for the pathway. With this finding in mind, it must be noted that there is A3A+UGI combination (three of three transfections) was hy- a hierarchy to A3 editing: HBV genomes are vulnerable to editing peredited MYC DNA recovered (Fig. S3 A and E). When a lower by six hA3 enzymes (17) and mtDNA could be edited by five (Fig. temperature range was explored, DNA was recovered down to 2E). HPV DNA was hyperedited by the three Zinc-finger mono- 88 °C. Phenomenally edited sequences with up to ∼70% target domain deaminases A3A, A3C, and A3H, but nuDNA is vulnerable residues edited were recovered (Fig. S3B). Although both strands to A3A only. Hence, nuDNA editing cannot result from a non- were edited, slightly fewer plus-strand hypermutants were re- physiological encounter of nuDNA with A3A, say in a lysed cell, covered. There was considerable sequence heterogeneity, indicating because the other active A3 enzymes would be involved. multiple independent deamination events. Not surprisingly, the Although A3 enzymes are involved in catabolizing DNA, there degree of editing was less for 3DPCR products recovered at 92.3 was a broad continuum with lightly edited sequences occurring than at 91 °C (Fig. S3C). Indeed, sequences with one to three C->T alongside the hyperedited sequences (Fig. S3C). As 3DPCR transitions were recovered, suggesting that there is a very large selects highly edited over lightly edited DNA, the fraction of the spectrum from editing, essentially from a few to ∼70%. The 5′ di- latter is certainly underestimated (17). Obviously, cells with A3- nucleotide context was strongly in favor of TpC+CpC and biased edited genomes will be subject to the winnowing power of pu- against GpC+ApC (Fig. S3D). rifying selection, with few survivors. The crucial question is, de- Not surprisingly, A3A transfection of a 293T cells stably spite the highly deleterious mutant spectrum resulting from A3 expressing UGI resulted in hyperedited MYC DNA (Fig. S3B). editing, can minute numbers of such cells escape death? Nuclear and cytoplasmic fractions were made from P1, P2, P3, The answer may well be positive for several reasons. First, A1 and A3A ± UGI transfected cells according to a variety of dif- and AICDA transgenic mice generally develop cancers dictated by

ferent protocols. However, the fractions were insufficiently pure the tg promoter (33–35). Second, the explosion in cancer genomics CELL BIOLOGY to draw conclusions as to the site of nuDNA editing. As UNG is showing that thousands of GC->AT transitions characterize impacted the detectable levels of MYC editing, we attempted the acancergenome(36–38). Third, although attention is focused on inverse, transfection by both A3A and the nuclear form of hu- the massively edited mitochondrial and nuDNA sequences, chro- − − man UNG, UNG2. As P3 gave the highest A3A genes expression mosomal DNA from a majority of P1 to P3 cells (ung / cell lines) (Fig. S2A), it and 293T-UGI cells were transfected with and contains uracil, as shown by the comet assay (28). As A3 genes are without UNG2 (29). For both P3 and 293T-UGI cells, less edited up-regulated by inflammatoryenvironments(4,5,7,8,17),itseems MYC DNA was recovered compared with the A2-negative con- that A3 deaminases will provide a recurrent source of genomic trol, reinforcing the notion that observed levels of editing reflect stress, particularly for persistent infections like viral hepatitis. a dynamic involving UNG (Fig. S2 B and C). In conclusion, there is far more cytidine deamination of hu- In an analogous fashion, the PCR/3DPCR approach was ap- man genomes than hitherto suspected. Indeed, the notion of plied to a region spanning most of exon 8 and part of intron 9 of spontaneous cytidine deamination may well need revisiting. The TP53. Like MYC, hypermutated DNA was obtained only from mutation load generated by hypermutation is incompatible with the A3A+UGI cotransfection (Fig. S4A). The data are highly replication, indicating an unsuspected mechanism of DNA ca- comparable with massively edited sequences harboring up to tabolism. The findings reveal a physiological role for the A3 59% of mutated cytidine residues, with the exception that only enzymes outside of the antiviral paradigm. Clearly, several A3 minus-strand hypermutants were found (Fig. S4B). A3A editing enzymes can function as human DNA mutators, and APO- spanned both exon 8 and intron 9 without discrimination. Editing BEC3A, like AICDA, becomes a serious candidate for nudging was once again biased in favor of TpC+CpC (Fig. S4C). genomes down the long and winding road to cancer. Discussion Materials and Methods The antiviral roles hitherto ascribed to A3 cytidine deaminases Samples. All of the liver materials have been previously described (17). The fit well with the observation that numerous human A3 genes study was approved by an institutional human research review board (RBM were up-regulated by interferons (4, 5, 7, 8). It turns out that this 2005–019). Informed consent was obtained for each patient. The PBMC activity represents just one aspect, the present data revealing an samples were either from healthy anonymous donors or obtained by written informed consent from sperm donors, following approval by local ethics unsuspected pathway for DNA catabolism of both human committees (Comités Consultatifs de Protection des Personnes dans la genomes initiated by cytidine editing, with A3A being particu- Recherche Biomédicale N°98353, and Comité de protection des personnes, larly important for nuDNA editing. UNG impacts the observed number AC-2009–886 Germéthèque) and the institutional review board levels of editing, but DNases may also be part of the equation, (Centre de Recherche de Biochimie 2003.6). − − and follows from the greater editing frequencies in ung / cells. Accordingly, there is more A3 editing than detected in normal Cells. One million HeLa cells were seeded in six-well plates and transfected 24 samples. Given the mutation load, A3 hyperedited nuDNA is h later by 3 μg of total plasmid using Fugene6 (Roche). Three days later, cells synonymous with cell death, the phenomenal loss of information were washed in cold PBS, covered by 1 mL of 10 mM Tris.HCl pH7.4, 2 mM making the process irreversible. MgCl2, and put on ice for 5 min, after which they were incubated with 0.5% fi fi Any hypothesis concerning mtDNA or nuDNA editing has to ( nal) Triton X-100 for 5 min on ice. Lysis was con rmed by microscopy. Cells > were subsequently incubated with 1 U/μL DNaseI (Roche) for 1 h at 37 °C, address a sequence with 50% of edited cytidines. To date, after which DNA was extracted as per usual. editing of this sort is found within the close confines of retroviral μ capsid structures, where A3 concentrations are in the 20- to 200- M Confocal Microscopy. Approximately 105 HeLa cells were transfected in 24- range, phenomenal enzyme concentrations by any measure, which well plates with C-terminal V5-tagged A3 constructs. At 72 h post- are essentially the result of capsid volumes in the zeptolitre range transfection, cells were incubated with 400 nM Mitotracker Deep Red FM (30, 31). In contrast, A3G hyperediting of ssDNA in vitro pla- (Invitrogen) for 40 min at 37 °C. After PBS washing, cells were fixed in 50/50

Suspène et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 methanol/acetone for 20 min at −20 °C. The anti-V5 antibody (Invitrogen) ACKNOWLEDGMENTS. We thank Drs. Carlo Battiston, Vincenzo Mazzaferro, was incubated at 1/200 for 1 h at room temperature, followed by incubation Kenneth McElreavey, Marie-Noëlle Ungeheuer, and Vesna Mellon for human with a fluoresceine-conjugated secondary antibody for 1 h at room tem- DNA samples, Prof. Wolfram Gerlich for tupaia liver samples, Dr. Philippe Blancou for horse and goat blood, Dr. Francina Langa Vives for the purified perature. Slides were stained with DAPI and mounted with Vectashield. mouse oocytes, Dr. Anne Durandy for the five Epstein Barr virus-transformed Confocal Imaging was performed using a Zeiss AxioImagerZ2 LSM700. B-cell lines and P1 DNA, Dr. Vincenzo Di Bartolo for the WTI and A3C1 cell lines, Pascal Roux for help with confocal microscopy, and Christophe Rusniok PCR. All DNAs were extracted using the Epicentre kit. All amplifications were for providing invaluable help with bioinformatics. This work is supported in performed using first-round standard PCR followed by nested 3DPCR (21) part by a postdoctoral fellowship from l’Association pour la Recherche sur le (Table S3). PCR was performed with 2.5 U Taq (Bioline) or 2.5 U Pfu (Stra- Cancer (to R.S.); a graduate fellowship from La Ligue contre le Cancer (to M.-M.A.); the Pasteur Foundation through its Zuccaire Internship Program tagene) DNA polymerase per reaction. PCR products were cloned using the (G.E.); and Grants from the Institut Pasteur, Agence Nationale de Recherches TOPO vector and sequencing was outsourced to Cogenics. Detailed in- sur le SIDA, Agence Nationale de Recherches, Institut National de la Santé et formation on PCR, 3D-PCR and real time quantitative PCR are provided in SI de la Recherche Médicale, and the Centre National de la Recherche Scientifi- Materials and Methods. que. The Unit is “Equipe labelisée LIGUE 2010.”

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