The nucleotide composition of microsatellites impacts both replication fidelity and mismatch repair in human colorectal cells

Introduction: Microsatellite instability is a key mechanism of colon carcinogenesis. We have previously studied mutations within a (CA)13 microsatellite using an EGFP-based reporter-assay that allows the distinction of replication errors and mismatch repair activity. Here we utilize this assay to compare mutations of mono- and dinucleotide repeats in human colorectal cells. Methods: HCT116 and HCT116+chr3 cells were stably transfected with EGFP-based plasmids harboring A10, G10, G16, (CA)13, and (CA)26 repeats. EGFP-positive mutant fractions were quantitated by flow cytometry, mutation rates were calculated, and the mutant spectrum was analyzed by cycle sequencing. Results: EGFP fluorescence pattern changed with the microsatellite's nucleotide sequence and cell type and clonal variations were observed in mononucleotide repeats. Replication errors (as calculated in HCT116) at A10 repeats were 5 to 10-fold higher than in G10, G16 were 30-fold higher than G10, and (CA) 26 were 10-fold higher than (CA)13. The mutation rates in hMLH1-proficient HCT116+chr3 were 30 to 230-fold lower than in HCT116. Mismatch repair was more efficient in G16 than in A10 clones leading to a higher stability of poly-G tracts. Mutation spectra revealed predominantly 1-unit-deletions in A10, (CA)13, and G10 and 2-unit-deletions or 1-unit-insertion in (CA)26. Discussion: These findings indicate that both replication fidelity and mismatch repair are affected by the microsatellite's nucleotide composition.