Adenosine Deaminase-Like Gene-Carried Lentivirus Toolkit for Identification of DNA N6-Methyladenine Origins

Abstract

Post-replicative DNA N⁶-methyladenine (pr6mdA) can form via bona fide methylase-catalyzed adenine methylation, playing a pivotal role in embryonic development and other biological processes. Surprisingly, pre-methylated adenine can be erroneously incorporated into DNA as misincorporated N6-methyladenine (i6mdA) via DNA polymerase-mediated replication. Despite pr6mdA and i6mdA sharing identical chemical structures, their biological functions diverge significantly, presenting a substantial challenge in distinguishing between the two. Here, for the first-time, it is exploited that the adenosine deaminase-like (Adal) protein and a corresponding activity-null mutant to construct an Adal lentivirus toolkit. With this newly designed toolkit, both pr6mdA and i6mdA can be identified and quantified simultaneously. The presence of 6mdA in the bone marrow cells of mice is shown, with its levels serving as indicators for growth with age, probably reflecting the cellular stress-caused changes in RNA decay, nucleotide pool sanitation, and transcription. Collectively, a powerful toolkit to advance understanding of both pr6mdA and i6mdA is demonstrated.

Keywords: DNA N6‐methyladenine; UHPLC‐MS/MS; adenosine deaminase‐like protein; isotope labeling.

Figure 1

Selection of Adal protein mutant as a control for toolkit identifying 6mdA origins. a) The key sites of mouse Adal (UniProt ID: Q80SY6). Adal H207A mutation site is marked with black triangle symbol. b) Flow diagram of the catalytic activity assay for purified MBP‐ Adal WT and mutant H207A. c–f) Quantification of substrates (6mdAMP c) and m6AMP e)) and products (dIMP d) and rIMP f)) upon deamination reaction mediated by purified MBP‐ Adal WT and mutant H207A. n = 2 independent biological replicates. ND: not detectable.

Scheme 1

Construction of Adal lentivirus toolkit. We amplified Adal WT CDS from cDNA reverse transcribed from total RNA of mES cells, and used point mutation to gain Adal H207A CDS. Then, we inserted CDS of Adal WT and mutant H207A into the pLVX‐EF1α‐IRES‐puro lentivirus vector plasmids. After transfecting lentivirus vectors plasmids into HEK‐293T cells, we obtained the Adal lentivirus toolkit for the detection of two types of 6mdA. Empty lentivirus served as a negative control.

Figure 2

Complete elimination of DNA i6mdA misincorporation by Adal lentivirus toolkit. a) Flow diagram of assessing the 6mdA misincorporation by Adal lentivirus toolkit. b) Schematic illustration of Adal eliminating isotope‐labeled i6mdA. c) The western blot analysis of overexpressed Adal in mES cells. d,e) UHPLC‐MS/MS chromatograms d) and quantification e) of [D₃]−6mdA in mES cells that overexpressed Adal. All mES cells were treated with 1 µM [D₃]‐m6rA for 24 h. f) The western blot analysis of overexpressed Adal in HEK‐293T. g,h) UHPLC‐MS/MS chromatograms g) and quantification h) of [D₃]−6mdA in Adal ‐overexpressed HEK‐293T cells. All HEK‐293T cells were treated with 5 µM [D₃]‐m6rA for 24 h. gDNA: genomic DNA. EV: empty vector. WT: wild‐type. Standard*: non‐labeled Standard. Error bars are S.D. (n = 3 independent biological replicates). ND: not detectable.

Figure 3

Methylase‐generated pr6mdA can be retained as assessed by Adal lentivirus toolkit. a) Flow diagram of assessing pr6mdA by Adal lentivirus toolkit. b) Schematic illustration of isotope‐labeled pr6mdA generation by activity‐reduced methylase Dam V181N. c) The western blot analysis and quantification of HA‐tagged Dam V181N overexpression in Adal lentivirus toolkit‐overexpressed HEK‐293T cells. Protein relative density was normalized to Adal WT. d,e) UHPLC‐MS/MS chromatograms d) and quantification e) of [D₃]−6mdA in HEK‐293T cells that simultaneous overexpressed Dam V181N and Adal. All HEK‐293T cells were treated with 30 µg mL⁻¹ [D₃]‐L‐methionine ([D₃]‐Met). gDNA: genomic DNA. EV: empty vector. WT: wild‐type. Standard*: non‐labeled Standard. Error bars are S.D. (n = 3 independent biological replicates). ND: not detectable.

Figure 4

Identification and quantification of i6mdA in C2C12 and NIH3T3 cells by Adal lentivirus toolkit. a) Flow diagram of identifying the origin of 6mdA in C2C12 and NIH3T3 cells by Adal lentivirus toolkit. b) The western blot analysis of overexpressed Adal in C2C12 cells. c,d) UHPLC‐MS/MS chromatograms c) and quantification d) of labeled 6mdA in Adal‐overexpressed C2C12 cells. All C2C12 cells were treated with 20 µM [¹⁵N₅]‐rA for 36 h. e) The western blot analysis of overexpressed Adal in NIH3T3 cells. f,g) UHPLC‐MS/MS chromatograms f) and quantification g) of labeled 6mdA in Adal‐overexpressed NIH3T3 cells. All NIH3T3 cells were treated with 20 µM [¹⁵N₅]‐rA for 36 h. gDNA: genomic DNA. EV: empty vector. WT: wild‐type. Standard*: non labeled Standard. Error bars are S.D. (n = 3 independent biological replicates). ND: not detectable.

Figure 5

MEL cell differentiation promotes the misincorporation of i6mdA. a) Flow diagram of identifying the type of 6mdA in MEL cells with Adal lentivirus toolkit. b) The western blot analysis of overexpressed Adal in MEL cells. c,d) UHPLC‐MS/MS chromatograms c) and quantification d) of [D₃]−6mdA in Adal‐overexpressed MEL cells. All MEL cells were treated by 30 µg mL⁻¹ [D₃]‐L‐methionine ([D₃]‐Met) during differentiation experiment. EV: empty vector. WT: wild‐type. Error bars are S.D. (n = 3 independent biological replicates).

Figure 6

DNA 6mdA in mouse BMCs and its correlation with age. a) Flow diagram of isolating BMCs from C57BL/6J mice of different ages. b) Flow cytometry sorting erythroblasts using antibodies against Ter119, CD71 of BMCs isolated from C57BL/6J male mice of different ages. c) UHPLC‐MS/MS quantification of 6mdA of total BMCs, Ter119+CD71+ BMCs, and Ter119‐ BMCs separated from C57BL/6J male mice of different ages. Error bars are S.D. (n = 3 independent biological replicates). d) The relationship between genomic 6mdA level and age in mouse total BMCs. A simple linear regression analysis was performed. Linear regression line, R‐squared, and p‐value were shown.