Abstract
The activin type II receptor (ACVR2) contains two identical microsatellites in exons 3 and 10, but only the exon 10 microsatellite is frameshifted in mismatch repair (MMR)-defective colonic tumors. The reason for this selectivity is not known. We hypothesized that ACVR2 frameshifts were influenced by DNA sequences surrounding the microsatellite. We constructed plasmids in which exons 3 or 10 of ACVR2 were cloned +1 bp out of frame of enhanced green fluorescent protein (EGFP), allowing –1 bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells, and subsequent non-fluorescent cells were sorted, cultured and harvested for mutation analysis. We swapped DNA sequences flanking the exon 3 and 10 microsatellites to test our hypothesis. Native ACVR2 exon 3 and 10 microsatellites underwent heteroduplex formation (A7/T8) in hMLH1−/− cells, but only exon 10 microsatellites fully mutated (A7/T7) in both hMLH1−/− and hMSH6−/− backgrounds, showing selectivity for exon 10 frameshifts and inability of exon 3 heteroduplexes to fully mutate. Substituting nucleotides flanking the exon 3 microsatellite for nucleotides flanking the exon 10 microsatellite significantly reduced heteroduplex and full mutation in hMLH1−/− cells. When the exon 3 microsatellite was flanked by nucleotides normally surrounding the exon 10 microsatellite, fully mutant exon 3 frameshifts appeared. Mutation selectivity for ACVR2 lies partly with flanking nucleotides surrounding each microsatellite.
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Abbreviations
- TGFBR2:
-
transforming growth factor β receptor 2
- ACVR2:
-
activin type II receptor
- MMR:
-
mismatch repair system
- MSI:
-
microsatellite instability
- IDL:
-
insertion deletion loop
- MR:
-
mutation resistant
- IF:
-
in frame of EGFP
- OF:
-
out of frame of EGFP
- EGFP:
-
enhanced green fluorescent protein
- CRC:
-
colorectal cancer
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Acknowledgements
This study was supported by the United States Public Health Service (DK067287 to JMC), the UCSD Digestive Diseases Research Development Center (DK080506), and the VA Research Service (Merit Review Award to JMC). We thank the support of Moores UCSD Cancer Center Flow Cytometry and DNA Sequencing Shared Resources.
Author contributions: Chung H and Carethers JM designed the research; Chung H, Young DJ, Lopez CG, Lai J, Holmstrom J, Cabrera BL and Ream-Robinson D performed the research; Chung H, Lopez CG and Carethers JM analyzed data; and Chung H and Carethers JM wrote the paper. All authors have approved the final version of this paper.
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Chung, H., Lopez, C., Young, D. et al. Flanking sequence specificity determines coding microsatellite heteroduplex and mutation rates with defective DNA mismatch repair (MMR). Oncogene 29, 2172–2180 (2010). https://doi.org/10.1038/onc.2009.508
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DOI: https://doi.org/10.1038/onc.2009.508