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Proc Natl Acad Sci U S A. Mar 22, 2011; 108(12): 4858–4863.
Published online Mar 2, 2011. doi:  10.1073/pnas.1009687108
PMCID: PMC3064337
Cell Biology

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

Abstract

The human APOBEC3 (A3A–A3H) locus encodes six cytidine deaminases that edit single-stranded DNA, the result being DNA peppered with uridine. Although several cytidine deaminases are clearly restriction factors for retroviruses and hepadnaviruses, it is not known if APOBEC3 enzymes have roles outside of these settings. It is shown here that both human mitochondrial and nuclear DNA are vulnerable to somatic hypermutation by A3 deaminases, with APOBEC3A standing out among them. The degree of editing is much greater in patients lacking the uracil DNA-glycolyase gene, indicating that the observed levels of editing reflect a dynamic composed of A3 editing and DNA catabolism involving uracil DNA-glycolyase. Nonetheless, hyper- and lightly mutated sequences went hand in hand, raising the hypothesis that recurrent low-level mutation by APOBEC3A could catalyze the transition from a healthy to a cancer genome.

The human APOBEC3 (A3) locus encodes seven expressed genes (A3A–A3H), of which six are demonstrably cytidine deaminases with specificity for single-stranded DNA (ssDNA) (1). The A3 locus arose in placental mammals by duplication of AICDA and subsequent expansion (2). Activation-induced cytidine deaminase (AICDA) is presently the only enzyme known to induce mutations in the human genome and initiates class-switch recombination and somatic hypermutation of rearranged Ig genes (3). Several human A3 genes are up-regulated in inflammatory settings by type I and II interferons, although activation depends to some degree on the cell type (48). Given this finding, it is not surprising that A3F and A3G are involved in the restriction of retroviruses and hepadnaviruses where nascent single-stranded cDNA remains vulnerable until double-strand synthesis is completed (916). In late stages of hepatitis B virus- (HBV) associated cirrhosis there was significant up-regulation of four to five A3 genes, with A3G being the major restriction factor able to edit up to 35% of HBV genomes (17).

DNA viral genomes, such as human papillomavirus (HPV), can also undergo editing both in vivo and in tissue culture, presumably during transcription or replication (18). Three A3 deaminases, A3A, A3C, and A3H could hyperedit HPV genomes ex vivo, suggesting that the phenomenon was occurring in the nucleus because the strictly cytoplasmic deaminases, A3F and A3G, did not edit transfected HPV DNA (18). Along with a recent article showing that A3A could edit transfected EGFP plasmid DNA (7), they highlight the potential of A3 enzymes to edit foreign DNA. The presence of uridine in cellular DNA is read as a danger signal, the uracil base resulting from cytidine deamination being subject to excision, particularly by uracil DNA-glycosylase (UNG), the nuclear form (UNG2) being far more active than its mitochondrial counterpart (UNG1). Uracil excision followed by abasic endonuclease cleavage of the DNA strand can lead to repair or degradation. Small fragments of nuclear and mitochondrial DNA (mtDNA) can be found in the cytoplasm, which trigger a number of cytoplasmic DNA sensor molecules and are associated with DNA degradation (19, 20).

As there are six human A3 enzymes, all with ssDNA specificity, the question arises as to whether they can edit host-cell DNA. Given the high mtDNA copy number per cell, we explored the possibility that a fraction of mtDNA might be vulnerable to A3 editing, especially in inflamed tissues where there is demonstrable up-regulation of A3 genes (17). We show here that a small fraction of human mitochondrial genomes are phenomenally edited by APOBEC3 deaminases in the cytoplasm. It transpires that nuclear DNA (nuDNA), notably segments of MYC and TP53, can be hyperedited by APOBEC3A. For both mitochondrial and nuDNA, editing reflects a dynamic between A3 activity and DNA degradation involving UNG.

Results

APOBEC3 Hypermutation of mtDNA.

A nested PCR/3DPCR approach was taken to the amplification of mtDNA from 36 DNA samples spanning alcoholic, HBV+, HBV+HCV+, and HCV+ cirrhosis (17, 21). A region corresponding to part of MT-COI at ~6,200 bp was chosen, as it maps within the large single-stranded replication intermediate. Mitochondrial DNA could be recovered at temperatures as low as 82.2 °C from 17 of 36 (~47%) samples tested, much lower than for a control liver or cloned MT-COI DNA (Td~86 °C) (Fig. 1 A and B). Molecular cloning and sequencing of the PCR products proved that they represented bona fide cytidine-edited mtDNA, with both strands being vulnerable (Fig. 1C and Fig. S1A), although minus-strand hypermutants were generally more abundant (shown as G->A hypermutants with respect to the plus strand). Although minus-strand editing was generally in the 30 to 80% range, occasionally a sequence with up to 98% of targets could be edited (Fig. 1 C and D).

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 ...

As A3 and AICDA catalyzed cytidine deamination is particularly sensitive to the 5′ base, a bulk dinucleotide analysis was performed for minus- and plus-strand hypermutants (Fig. 1 E and F). The strong biases for TpC and CpC among minus-strand hypermutants are hallmarks of APOBEC3 editing (11, 15, 22). For plus-strand editing, the clear preference for CpC is a hallmark of A3G (Fig. 1F), no matter the template (11). These slightly different patterns probably reflect the greater C content of the plus strand generating more oligoC tracts, which are known hotspots for A3G. A clonal analysis of the dinucleotide context for three liver samples showed that all edited mtDNA sequences mapped in the region typical of A3 deaminases (Fig. 1G), meaning that there was not also a handful of AICDA-edited sequences, which would show up in the GpC+ApC region, hidden among a majority of A3-edited sequences.

Mitochondrial DNA Editing in Peripheral-Blood Mononuclear Cells.

Virus-infected tissues can be infiltrated by leukocytes, but numerous studies have described A3 expression in resting and activated leukocytes (5, 6, 23). To explore this issue, total DNA from 11 Ficoll-purified peripheral-blood mononuclear cells (PBMCs) and 3 Epstein Barr virus (EBV)-transformed PBMCs was analyzed (Table S1). In all cases, 3DPCR-recovered edited mtDNA way below the reference temperature of ~86 °C (Fig. 2A). This finding is interesting in that the samples spanned a broad age range: from a 3-wk-old child without pathology to nonagenerians (Table S1). Sequence analysis confirmed A3 hyperediting (i.e., there was a strong bias in favor of TpC and CpC, typical of A3 deaminases). Citrate-treated blood from two donors was Ficoll-fractionated using antibody-coated magnetic beads to purify CD4+ and CD8+ T cells, CD14+ monocytes, CD15+ neutrophils, CD16+ eosinophils+basophils, CD19+ B cells, and CD56+ natural killer cells. MT-COI editing was evident for all fractions, the neutrophils showing the greatest degree of editing: that is, 3DPCR products were recovered as low as 82.2 °C (Fig. 2B). Sequencing showed that cytidine deamination was concentrated in TpC and CpC motifs for all cell subsets, indicating that the same A3, or ensemble of A3 enzymes, was responsible (Fig. 2D). Numerous human cell lines are known to express A3 genes (1) and eight of eight cell lines tested positive for hyperedited mtDNA (Fig. 2C). To get an independent idea of the frequencies of mtDNA editing in PBMCs, we sequenced ~1,000 clones from standard PCR for two samples (AS0488 and AS2017) (Table S1) yet did not find a single hypermutated sequence indicating hypermutant frequencies of <10−3.

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. ...

Mitochondrial DNA Editing Occurs Outside of the Mitochondrion.

The intracellular localization of transfected A3-V5 tagged constructs was analyzed by confocal microscopy using Mitrotracker deep red. Despite extensive screening, we were unable to find any colocalization of any A3 with the mitochondrial network, which would show up as strong diagonals (Fig. 2E), suggesting that editing occurred in the cytoplasm. To explore this theory, HeLa cells were treated with 0.5% Triton X-100 and incubated with 1 μg/mL of DNaseI for 1 h, which removed the hyperedited mtDNA yet left a strong normal mtDNA signal (Fig. 2F). As DNase treatment alone did not reduce the edited DNA signal, this rules out extracellular mtDNA as a source for the signal (Fig. 2F). If hyperediting occurred outside of the mitochondrion, it should not be subject to DNA repair. Like all archaeabacterial polymerases, Pfu is unable to amplify DNA harboring dU (24). We performed 3DPCR on first round PCR mtDNA amplified at 95 °C using Pfu. This strategy failed to recover any genomes beyond the 85.4 to 86 °C benchmark temperature for unedited mtDNA (Fig. 2F). These findings show that A3 editing of mtDNA occurs outside of the mitochondrion and goes uncorrected.

The high levels of hyperedited mtDNA in established cell lines has thwarted detailed analysis of the phenomenon by transfection experiments. Only for A3A transfections, and to a lesser extent A3G, could a clear signal over background be found (Fig. 2F). Uracil-DNA glycosylase inhibitor (UGI) is a small protein from a Bacillus subtilis bacteriophage that can inhibit mammalian UNG, a crucial enzyme involved in excising uracil from DNA (25). When A3A or A3G were cotransfected with the UGI plasmid, mtDNA recovered at temperatures as low as 81 °C (Fig. 2F).

Given this finding, we next turned to EBV transformed B-cell lines from three ung−/− individuals [P1, P2, and P3 (26)]. Hyperedited mtDNA was detected for all three samples down to 81 °C, confirming the results had with UGI (Fig. 2F). To determine A3 expression levels, real-time quantitative PCR was performed for P1, P2, and P3, as well as two ung+ control EBV-transformed cell lines, Z (AICDA−/−) and T (Fig. S2A). Data were normalized to the expression levels of four invariable reference genes (CLK2, GUSB, HMBS, TBP). Although A3 expression was in general similar, A3A showed some variation among the samples (Fig. S2A). Analysis of total PBMC DNA from one of these individuals (P1) yielded the same result as the cell line, ruling out any influence from EBV on the observation. For P2 DNA, the frequency of mtDNA hypermutants in standard PCR products was assessed by deep sequencing. Approximately 0.5% were edited, greater than for PBMC DNA from normal individuals (vide supra). This finding translates to ~25 per cell, assuming ~5 K mtDNA genomes per cell.

Limiting-dilution of PBMCs and purified CD4 cells from patient ABD2 were performed in triplicate, the hyperedited 3DPCR signal being lost between 1,000 and 300 cells (f ≥ 10−3). A similar triplicate titration of P2 cells resulted in a loss of signal between 50 and 10 cells (f ≥ 2 × 10−2). Given 3DPCR as readout, these frequencies are underestimates. For P2 cells where the frequencies of cells harboring edited mtDNA and bulk hyperedited mtDNA are known, it can be concluded that a sizeable fraction of cells harbors a small fraction of hyperedited mtDNA sequences.

All of the above analyses concerned editing of the the MT-COI 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 by 3DPCR. Like MT-COI, the former maps to the ssDNA replication intermediate but MT-ND2 mapped to the dsDNA region. Both DNA strands of MT-CYB and MT-ND2 were heavily edited; a selection from P2 cells is shown in Fig. S1B.

Although A3A and A3G can edit mtDNA, this doesn't exclude other A3 enzymes that could be individually, or together, responsible for the high background. To address this question we used the QT6 quail cell line that has never shown an endogenous cytidine-editing background for retroviruses, presumably because the avian lineage does not encode A1 or A3 othologs (11). QT6 cells were transfected by numerous APOBEC plasmids. The PCR/3DPCR approach recovered hyperedited quail mtDNA for 5 of 11 human cytidine deaminases, notably A3A, A3C, A3F, A3G, and A3H; the murine A1 and A3 enzymes were also capable of editing QT6 mtDNA (Fig. 2G).

Given this result, it is possible that mtDNA editing may occur beyond the primate world, with its seven-gene A3 locus. For other mammals, the A3 gene number presently ranges from one (mice, rats, pigs), to three (cats) and six (horses) (27). In contrast, birds, reptiles, and fish do not encode an A3 gene. Edited mtDNA was recovered from tree shrew hepatocyte cultures (Tupaia belangeri belongs to Scandentia, the closest order to Primates) as well as horse and goat PBMCs (Table S2). In all cases, 5′ dinucleotide analysis revealed a penchant for editing within TpC and CpC, typical of A3 enzymes. Mouse DNA from numerous tissues was systematically negative for mtDNA editing (Table S2). These findings show that the phenomenon of mtDNA editing is not uniform across placental mammals.

A3 Editing of the Human nuDNA.

As the A3A, A3C, and A3H enzymes in particular can locate to the nucleus, we tested the possibility that, occasionally, nuDNA might be vulnerable to A3 editing. MYC is particularly mutated in leukemias and lymphomas, and the tumor-suppressor gene TP53 is mutated in ~50% of cancers, with exon 8 encoding many hotspots (http://p53.free.fr). Several of the human cirrhosis and PBMC samples where mtDNA editing was in evidence were screened using primers for MYC exon 2 and TP53 exon 8/intron 9. We were unable to recover massively hypermutated MYC or TP53 sequences.

Given the impact of UNG on mtDNA editing (Fig. 2F), we turned to the three human ung−/− cell lines [P1, P2, and P3 (26, 28)]. Hyperedited MYC DNA was recovered as low as 91 °C from all three cell lines, as well as from P1 PBMC DNA (Fig. 3A). Sequencing revealed a range of editing, from 1 to 38 C->T transitions per strand (Fig. 3B), with a strong penchant for TpC and CpC (Fig. 3 C–E). The frequency of hyperdited MYC sequences can be estimated from the number of unique sequences derived from 0.5-μg input DNA (~150 K copies). As ~30 to 50% of sequences were identical, the number of distinct sequences is in the range of the number of unique sequences. Hence, the hyperedited MYC frequencies are in the range of 20/150 K ~10−4. At such low frequencies, correlations with macroscopic, such as bulk transciption, levels are going to be fraught with problems.

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 ...

To explore nuDNA editing experimentally, we transfected HeLa cells with individual A3-V5 constructs ±UGI and sought hyperedited MYC DNA at 72 h posttransfection. Only for the A3A+UGI combination (three of three transfections) was hyperedited MYC DNA recovered (Fig. S3 A and E). When a lower temperature range was explored, DNA was recovered down to 88 °C. Phenomenally edited sequences with up to ~70% target residues edited were recovered (Fig. S3B). Although both strands were edited, slightly fewer plus-strand hypermutants were recovered. There was considerable sequence heterogeneity, indicating multiple independent deamination events. Not surprisingly, the degree of editing was less for 3DPCR products recovered at 92.3 than at 91 °C (Fig. S3C). Indeed, sequences with one to three C->T transitions were recovered, suggesting that there is a very large spectrum from editing, essentially from a few to ~70%. The 5′ dinucleotide context was strongly in favor of TpC+CpC and biased against GpC+ApC (Fig. S3D).

Not surprisingly, A3A transfection of a 293T cells stably expressing UGI resulted in hyperedited MYC DNA (Fig. S3B). Nuclear and cytoplasmic fractions were made from P1, P2, P3, and A3A ± UGI transfected cells according to a variety of different protocols. However, the fractions were insufficiently pure to draw conclusions as to the site of nuDNA editing. As UNG impacted the detectable levels of MYC editing, we attempted the inverse, transfection by both A3A and the nuclear form of human UNG, UNG2. As P3 gave the highest A3A genes expression (Fig. S2A), it and 293T-UGI cells were transfected with and without UNG2 (29). For both P3 and 293T-UGI cells, less edited MYC DNA was recovered compared with the A2-negative control, reinforcing the notion that observed levels of editing reflect a dynamic involving UNG (Fig. S2 B and C).

In an analogous fashion, the PCR/3DPCR approach was applied to a region spanning most of exon 8 and part of intron 9 of TP53. Like MYC, hypermutated DNA was obtained only from the A3A+UGI cotransfection (Fig. S4A). The data are highly comparable with massively edited sequences harboring up to 59% of mutated cytidine residues, with the exception that only minus-strand hypermutants were found (Fig. S4B). A3A editing spanned both exon 8 and intron 9 without discrimination. Editing was once again biased in favor of TpC+CpC (Fig. S4C).

Discussion

The antiviral roles hitherto ascribed to A3 cytidine deaminases fit well with the observation that numerous human A3 genes were up-regulated by interferons (4, 5, 7, 8). It turns out that this activity represents just one aspect, the present data revealing an unsuspected pathway for DNA catabolism of both human genomes initiated by cytidine editing, with A3A being particularly important for nuDNA editing. UNG impacts the observed levels of editing, but DNases may also be part of the equation, and follows from the greater editing frequencies in ung−/− cells. Accordingly, there is more A3 editing than detected in normal samples. Given the mutation load, A3 hyperedited nuDNA is synonymous with cell death, the phenomenal loss of information making the process irreversible.

Any hypothesis concerning mtDNA or nuDNA editing has to address a sequence with >50% of edited cytidines. To date, editing of this sort is found within the close confines of retroviral capsid structures, where A3 concentrations are in the 20- to 200-μM range, phenomenal enzyme concentrations by any measure, which are essentially the result of capsid volumes in the zeptolitre range (30, 31). In contrast, A3G hyperediting of ssDNA in vitro plateaued at ~20% of cytidines deaminated at ~2 μM of enzyme (32), meaning that the hyperediting observed cannot be simply the chance encounter of free enzyme and target ssDNA in a permeablized or collapsed cell. The Triton-dependent DNase sensitivity of edited mtDNA supports this deduction. Hypermutated human DNA therefore implies compartmentalization or cofactors where ssDNA and A3 remain together, rather than permitted to drift apart, and so contributing to the efficiency of the catabolic pathway. With this finding in mind, it must be noted that there is a hierarchy to A3 editing: HBV genomes are vulnerable to editing by six hA3 enzymes (17) and mtDNA could be edited by five (Fig. 2E). HPV DNA was hyperedited by the three Zinc-finger monodomain deaminases A3A, A3C, and A3H, but nuDNA is vulnerable to A3A only. Hence, nuDNA editing cannot result from a nonphysiological encounter of nuDNA with A3A, say in a lysed cell, because the other active A3 enzymes would be involved.

Although A3 enzymes are involved in catabolizing DNA, there was a broad continuum with lightly edited sequences occurring alongside the hyperedited sequences (Fig. S3C). As 3DPCR selects highly edited over lightly edited DNA, the fraction of the latter is certainly underestimated (17). Obviously, cells with A3-edited genomes will be subject to the winnowing power of purifying selection, with few survivors. The crucial question is, despite the highly deleterious mutant spectrum resulting from A3 editing, can minute numbers of such cells escape death?

The answer may well be positive for several reasons. First, A1 and AICDA transgenic mice generally develop cancers dictated by the tg promoter (3335). Second, the explosion in cancer genomics is showing that thousands of GC->AT transitions characterize a cancer genome (3638). Third, although attention is focused on the massively edited mitochondrial and nuDNA sequences, chromosomal DNA from a majority of P1 to P3 cells (ung−/− cell lines) contains uracil, as shown by the comet assay (28). As A3 genes are up-regulated by inflammatory environments (4, 5, 7, 8, 17), it seems that A3 deaminases will provide a recurrent source of genomic stress, particularly for persistent infections like viral hepatitis.

In conclusion, there is far more cytidine deamination of human genomes than hitherto suspected. Indeed, the notion of spontaneous cytidine deamination may well need revisiting. The mutation load generated by hypermutation is incompatible with replication, indicating an unsuspected mechanism of DNA catabolism. The findings reveal a physiological role for the A3 enzymes outside of the antiviral paradigm. Clearly, several A3 enzymes can function as human DNA mutators, and APOBEC3A, like AICDA, becomes a serious candidate for nudging genomes down the long and winding road to cancer.

Materials and Methods

Samples.

All of the liver materials have been previously described (17). The study was approved by an institutional human research review board (RBM 2005–019). Informed consent was obtained for each patient. The PBMC samples were either from healthy anonymous donors or obtained by written informed consent from sperm donors, following approval by local ethics committees (Comités Consultatifs de Protection des Personnes dans la Recherche Biomédicale N°98353, and Comité de protection des personnes, number AC-2009–886 Germéthèque) and the institutional review board (Centre de Recherche de Biochimie 2003.6).

Cells.

One million HeLa cells were seeded in six-well plates and transfected 24 h later by 3 μg of total plasmid using Fugene6 (Roche). Three days later, cells were washed in cold PBS, covered by 1 mL of 10 mM Tris.HCl pH7.4, 2 mM MgCl2, and put on ice for 5 min, after which they were incubated with 0.5% (final) Triton X-100 for 5 min on ice. Lysis was confirmed by microscopy. Cells were subsequently incubated with 1 U/μL DNaseI (Roche) for 1 h at 37 °C, after which DNA was extracted as per usual.

Confocal Microscopy.

Approximately 105 HeLa cells were transfected in 24-well plates with C-terminal V5-tagged A3 constructs. At 72 h posttransfection, cells were incubated with 400 nM Mitotracker Deep Red FM (Invitrogen) for 40 min at 37 °C. After PBS washing, cells were fixed in 50/50 methanol/acetone for 20 min at −20 °C. The anti-V5 antibody (Invitrogen) was incubated at 1/200 for 1 h at room temperature, followed by incubation with a fluoresceine-conjugated secondary antibody for 1 h at room temperature. Slides were stained with DAPI and mounted with Vectashield. Confocal Imaging was performed using a Zeiss AxioImagerZ2 LSM700.

PCR.

All DNAs were extracted using the Epicentre kit. All amplifications were performed using first-round standard PCR followed by nested 3DPCR (21) (Table S3). PCR was performed with 2.5 U Taq (Bioline) or 2.5 U Pfu (Stratagene) DNA polymerase per reaction. PCR products were cloned using the TOPO vector and sequencing was outsourced to Cogenics. Detailed information on PCR, 3D-PCR and real time quantitative PCR are provided in SI Materials and Methods.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Drs. Carlo Battiston, Vincenzo Mazzaferro, Kenneth McElreavey, Marie-Noëlle Ungeheuer, and Vesna Mellon for human DNA samples, Prof. Wolfram Gerlich for tupaia liver samples, Dr. Philippe Blancou for horse and goat blood, Dr. Francina Langa Vives for the purified mouse oocytes, Dr. Anne Durandy for the five Epstein Barr virus-transformed 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 for providing invaluable help with bioinformatics. This work is supported in part by a postdoctoral fellowship from l'Association pour la Recherche sur le 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 (G.E.); and Grants from the Institut Pasteur, Agence Nationale de Recherches sur le SIDA, Agence Nationale de Recherches, Institut National de la Santé et de la Recherche Médicale, and the Centre National de la Recherche Scientifique. The Unit is “Equipe labelisée LIGUE 2010.”

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. R.S.H. is a guest editor invited by the Editorial Board.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1009687108/-/DCSupplemental.

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