Fig. 4: Principle and results of pulse-chase NAIL-MS experiments to determine the repair of ms²C in vivo.

a Principle of a pulse-chase NAIL-MS experiment. The bacteria are grown in unlabeled (n.l.) media before and after exposure to MMS (structure shown). After 1 h MMS exposure, the media is removed and fresh, [¹⁵N], [³⁴S] and [CD₃]-methionine containing media is added. Samples are drawn during the recovery time for tRNA isolation. b Formation of the nucleoside damage product ms²C (blue) and m¹A (black) per average tRNA after 20 mM MMS exposure. *Dashed lines: abundance of mod. (ms²C and m¹A) per all tRNAs (sum of original, unlabeled and new, [¹⁵N]-labeled transcripts). Solid lines: abundance of mod. (ms²C and m¹A) per original tRNA. c Abundance of mod. (ms²C in blue, m¹A in dark gray and s²C in light gray) per tRNA incubated with purified AlkB in vitro. The substrate tRNA is isolated from E. coli bacteria exposed to 20 mM MMS. d ms²C abundance of E. coli WT (black) in comparison to alkB deficient E. coli (ΔalkB, blue) after MMS stress (20 mM, 1 h) and during recovery in a pulse-chase NAIL-MS experiment, as described in a) and b). e s²C abundance of E. coli WT (black) in comparison to alkB deficient E. coli (ΔalkB, blue) after MMS stress (20 mM, 1 h) and during recovery. All experiments are from n = 3 biol. replicates and error bars reflect standard deviation. p-values from student t-test (equal distribution, two-sided): *p < 0.05 and **p < 0.01. Source data are provided as a Source Data file.