Both microsatellite length and sequence context determine frameshift mutation rates in defective DNA mismatch repair

It is generally accepted that longer microsatellites mutate more frequently in defective DNA mismatch repair (MMR) than shorter microsatellites. Indeed, we have previously observed that the A(10) microsatellite of transforming growth factor beta type II receptor (TGFBR2) frameshifts -1 bp at a faster rate than the A(8) microsatellite of activin type II receptor (ACVR2), although both genes become frameshift-mutated in >80% of MMR-defective colorectal cancers. To experimentally determine the effect of microsatellite length upon frameshift mutation in gene-specific sequence contexts, we altered the microsatellite length within TGFBR2 exon 3 and ACVR2 exon 10, generating A(7), A(10) and A(13) constructs. These constructs were cloned 1 bp out of frame of EGFP, allowing a -1 bp frameshift to drive EGFP expression, and stably transfected into MMR-deficient cells. Subsequent non-fluorescent cells were sorted, cultured for 7-35 days and harvested for EGFP analysis and DNA sequencing. Longer microsatellites within TGFBR2 and ACVR2 showed significantly higher mutation rates than shorter ones, with TGFBR2 A(13), A(10) and A(7) frameshifts measured at 22.38 x 10(-4), 2.17 x 10(-4) and 0.13 x 10(-4), respectively. Surprisingly, shorter ACVR2 constructs showed three times higher mutation rates at A(7) and A(10) lengths than identical length TGFBR2 constructs but comparably lower at the A(13) length, suggesting influences from both microsatellite length as well as the sequence context. Furthermore, the TGFBR2 A(13) construct mutated into 33% A(11) sequences (-2 bp) in addition to expected A(12) (-1 bp), indicating that this construct undergoes continual subsequent frameshift mutation. These data demonstrate experimentally that both the length of a mononucleotide microsatellite and its sequence context influence mutation rate in defective DNA MMR.