Role of TdT in the causation of FLT3-ITD and NPM1c mutations in AML. (A) In normal DNA replication, the daughter strand (gray) is a perfect complement of the template strand (black). (B) FLT3-ITDs can arise as a result of replication slippage during which the DNA polymerase resumes replication from an upstream position microhomologous with the end of the elongating daughter strand (red). (C) In approximately one-third of FLT3-ITDs, there is no visible microhomology. Instead, TdT is predicted to add nucleotides (blue) to the end of the daughter strand in order to provide the occult (or “missing”) microhomology (of at least 1 basepair) between the daughter and template strands. In this specific instance, a run of 5 Gs was added, only the final one of which was used for microhomology. The first 4 Gs then appear as a “filler” sequence in the final mutant DNA and also serve to keep the FLT3 mRNA reading frame open (the total number of additional nucleotides [21] is divisible by 3). (D) Similarly, a single T nucleotide is predicted to be added to the daughter strand during the formation of the common NPM1 mutation, a 4-bp tandem duplication. Borrow et al provide evidence that the extra nucleotides facilitating the formation of approximately one-third of FLT3-ITDs and almost all NPM1c mutations²  follow a pattern that strongly suggests illegitimate TdT activity. Panels B to D are derived from real examples of mutations depicted by Borrow et al. bp, basepair; nt, nucleotide. Professional illustration by Patrick Lane, ScEYEnce Studios.