Somatic Hypermutation of Human Mitochondrial and Nuclear DNA by APOBEC3 Cytidine Deaminases, a Pathway for DNA Catabolism
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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.