Aberrant DNA N6-methyladenine incorporation via adenylate kinase 1 is suppressed by ADAL deaminase-dependent 2'-deoxynucleotide pool sanitation

Intracellular decay of N6-methyladenine (m6A)-containing RNA potentially induces aberrant N6-methyl-2'-adenine (6mdA) misincorporation into DNA. Biophysically, misincorporated 6mdA may destabilize the DNA duplex in a manner similar to bona fide methylated 6mdA DNA, thereby affecting DNA replication and transcription. Utilizing heavy stable isotope labeling and ultrasensitive UHPLC-MS/MS assay, we demonstrate that intracellular m6A-RNA decay does not generate free 6mdA species, nor lead to any misincorporated DNA 6mdA in most mammalian cell lines tested, unveiling the existence of a sanitation mechanism that prevents 6mdA misincorporation. Depletion of deaminase ADAL increases the levels of free 6mdA species, concomitant with the presence of DNA-misincorporated 6mdA resulting from intracellular RNA m6A decay, suggesting that ADAL catabolizes 6mdAMP in vivo. Furthermore, we show that overexpression of adenylate kinase 1 (AK1) promotes 6mdA misincorporation, while AK1 knockdown diminishes 6mdA incorporation, in ADAL-deficient cells. We conclude that ADAL together with other factors (such as MTH1) contribute to 2'-deoxynucleotide pool sanitation in most cells, but compromised sanitation (as e.g., in NIH3T3 cells) and increased AK1 expression may facilitate aberrant 6mdA incorporation. This sanitation mechanism may provide a framework for maintenance of the epigenetic 6mdA landscape.

含有N6-甲基腺嘌呤（N6-methyladenine, m6A）的RNA在细胞内发生降解，可能会诱导异常的N6-甲基-2'-腺嘌呤（N6-methyl-2'-adenine, 6mdA）错误掺入DNA中。从生物物理层面来看，错误掺入的6mdA可通过与天然甲基化6mdA DNA相似的方式破坏DNA双链稳定性，进而影响DNA复制与转录过程。本研究采用重稳定同位素标记与超灵敏超高效液相色谱-串联质谱（UHPLC-MS/MS）分析技术，实验证实：在绝大多数受试哺乳动物细胞系中，细胞内携带m6A修饰的RNA降解既不会产生游离的6mdA物质，也不会引发DNA出现错误掺入的6mdA，这揭示了一类可阻断6mdA错误掺入的净化机制。脱氨酶ADAL的敲低会使游离6mdA物质的水平升高，同时伴随由细胞内RNA的m6A降解所导致的DNA错误掺入6mdA现象，这表明ADAL在体内可代谢6mdAMP。此外，在ADAL缺陷的细胞中，本研究发现腺苷酸激酶1（adenylate kinase 1, AK1）过表达会促进6mdA的错误掺入，而AK1敲低则会减少6mdA的掺入量。综上，本研究认为：在绝大多数细胞中，ADAL与其他因子（例如MTH1）共同参与2'-脱氧核苷酸池的净化维持；而当净化机制受损（如在NIH3T3细胞中）或AK1表达上调时，则可能会促进异常的6mdA掺入。该净化机制或可为表观遗传6mdA修饰谱的维持提供理论框架。