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

摘要: 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 these TGFbeta family receptors is abrogated in DNA Mismatch repair (MMR)-deficient tumors. How occur real time mutational rates human sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1-/-, hMSH6-/-, hMSH3-/-, MMR-proficient) to determine mutation rates. Plasmids were constructed which exon 3 10 ACVR2 cloned +1 bp out frame, immediately after the translation initiation codon an enhanced GFP (EGFP) gene, allowing -1 drive EGFP expression. Mutation-resistant plasmids by interrupting microsatellite sequences, preventing mutation. Stable established containing portions ACVR2, nonfluorescent cells sorted, cultured for 7-35 days, harvested flow cytometric detection sequencing specific points. revealed (A9 A7 ACVR2) fluorescent cells. Two distinct populations, M1 (dim, representing heteroduplexes) M2 (bright, full mutants) identified, fraction accumulating over time. hMLH1 deficiency 11 (5.91 x 10(-4)) 15 (2.18 times higher compared hMSH6 deficiency, respectively. The rate was approximately both than microsatellite. are dependent upon background.