Both microsatellite length and sequence context determine frameshift mutation rates in defective DNA mismatch repair
| Autor: | Dennis J. Young, Deena Ream-Robinson, Joy Holmstrom, Heekyung Chung, Claudia G. Lopez, John M. Carethers, Jenny F. Lai |
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| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
Mutation rate
Activin Receptors Type II Green Fluorescent Proteins Context (language use) Biology Protein Serine-Threonine Kinases DNA Mismatch Repair Frameshift mutation Exon Cell Line Tumor Genetics Humans Frameshift Mutation Molecular Biology Gene Genetics (clinical) Base Sequence Receptor Transforming Growth Factor-beta Type II General Medicine Articles Molecular biology Mutation (genetic algorithm) Microsatellite DNA mismatch repair Receptors Transforming Growth Factor beta Microsatellite Repeats |
| Popis: | 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 × 10 -4 , 2.17 × 10 -4 and 0.13 × 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. |
| Databáze: | OpenAIRE |
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