Genotoxic stress increases cytoplasmic mitochondrial DNA editing by human APOBEC3 mutator enzymes at a single cell level

Bianka Mussil (1) , Rodolphe Suspene (1) , Vincent Caval (1) , Anne Durandy (2) , Simon Wain-Hobson (1) , Jean Pierre Vartanian (1)

1 Rétrovirologie moléculaire - Molecular Retrovirology
2 IMAGINE - U1163 - Imagine - Institut des maladies génétiques

Human cells are stressed by numerous mechanisms that can lead to leakage of mitochondrial DNA (mtDNA) to the cytoplasm and ultimately apoptosis. this agonist DNA constitutes a danger to the cell and is counteracted by cytoplasmic DNases and APOBEC3 cytidine deamination of DNA. To investigate APOBEC3 editing of leaked mtDNA to the cytoplasm, we performed a PCR analysis of APOBEC3 edited cytoplasmic mtDNA (cymtDNA) at the single cell level for primary CD4 + T cells and the established P2 EBV blast cell line. Up to 17% of primary CD4 + T cells showed signs of APOBEC3 edited cymtDNA with ~50% of all mtDNA sequences showing signs of APOBEC3 editing-between 1500-5000 molecules. Although the P2 cell line showed a much lower frequency of stressed cells, the number of edited mtDNA molecules in such cells was of the same order. Addition of the genotoxic molecules, etoposide or actinomycin D increased the number of cells showing APOBEC3 edited cymtDNA to around 40%. These findings reveal a very dynamic image of the mitochondrial network, which changes considerably under stress. APOBEC3 deaminases are involved in the catabolism of mitochondrial DNA to circumvent chronic immune stimulation triggered by released mitochondrial DNA from damaged cells. The APOBEC3 (A3) locus encodes a series of seven genes encoding six functional endogenous cytidine deam-inases with substrate specificity for single stranded DNA (ssDNA) 1. They leave DNA peppered with uracil residues. This process is referred to as genetic editing as it occurs post replication. A3 enzymes leave a telltale editing signature in DNA: most A3 enzymes preferentially edit a cytidine residue in the context of 5′TpC with the exception of A3G, which prefers 5′CpC dinucleotides 2-6. The antiviral role of these A3 enzymes was initially highlighted by their impact on HIV and HBV replication 6-9 and the fact that several A3 genes can be up-regulated by interferon α 10-12. A3 can restrict the transposition of SINE and LINE retroelements 13,14 and mitochondrial DNA (mtDNA) in cell lines and tissues, all of which show the A3 editing signature-5′TpC and 5′CpC are preferentially deam-inated 15. For the latter, the target was cytoplasmic mtDNA (cymtDNA) in keeping with the observation that all A3 enzymes are unable to access the mitochondrial network 15. In addition, A3A and to a lesser extent A3B, were shown to target chromosomal DNA non-specifically, leading to hypermutated DNA with up to 70% of cyti-dine residues deaminated 15,16. A3A and A3B-induced editing occur predominantly on the lagging strand during DNA replication 17,18. Interestingly, A3A and A3B can edit 5-methylcytidine (5MeC) residues in ssDNA which makes sense given that 5MeC is found exclusively in the nucleus 16,19-21. Uracil bases in DNA are excised by uracil N-glycosidase (UNG) that initiates DNA damage responses, which can result in either DNA repair or catabolism.

Domaines: Biologie moléculaire

Bianka Mussil, Rodolphe Suspene, Vincent Caval, Anne Durandy, Simon Wain-Hobson, et al.. Genotoxic stress increases cytoplasmic mitochondrial DNA editing by human APOBEC3 mutator enzymes at a single cell level. Scientific Reports, 2019, 9 (1), pp.3109. ⟨10.1038/s41598-019-39245-8⟩. ⟨pasteur-02065267⟩