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Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as  TGFBR2  and  ACVR2 . As a consequence, signaling through these TGFβ family receptors is abrogated in DNA Mismatch repair (MMR)-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies ( hMLH1 −/−  ,  hMSH6 −/−  ,  hMSH3 −/−  , and MMR-proficient) to determine mutation rates. Plasmids were constructed in which exon 3 of  TGFBR2  and exon 10 of  ACVR2  were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP) gene, allowing a −1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of  TGFBR2  and  ACVR2 , and nonfluorescent cells were sorted, cultured for 7–35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a −1 bp frameshift mutation (A 9  in  TGFBR2  and A 7  in  ACVR2 ) in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes) and M2 (bright, representing full mutants) were identified, with the M2 fraction accumulating over time.  hMLH1  deficiency revealed 11 (5.91×10 −4 ) and 15 (2.18×10 −4 ) times higher mutation rates for the  TGFBR2  and  ACVR2  microsatellites compared to  hMSH6  deficiency, respectively. The mutation rate of the  TGFBR2  microsatellite was ∼3 times higher in both  hMLH1  and  hMSH6  deficiencies than the  ACVR2  microsatellite. The −1 bp frameshift mutation rates of  TGFBR2  and  ACVR2  microsatellite sequences are dependent upon the human MMR background.

Mutation Rates of TGFBR2 and ACVR2 Coding Microsatellites in Human Cells with Defective DNA Mismatch Repair