Flanking sequence specificity determines coding microsatellite heteroduplex and mutation rates with defective DNA mismatch repair (MMR)

Abstract

The activin type II receptor (ACVR2) contains 2 identical microsatellites in exon 3 and 10, but only the exon 10 microsatellite is frameshifted in 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 exon 3 or 10 of ACVR2 were cloned +1bp out-of-frame of EGFP, allowing −1bp frameshift to express EGFP. Plasmids were stably-transfected into MMR-deficient cells, subsequent non-fluorescent cells 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.

Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications

“…In absence of functional studies, discriminating between the genes that are true targets for colorectal MSI-driven mutagenesis from those whose mutations occur randomly and are left unrepaired due to the absence of MMR is not self-evident. High mutation prevalence in a given gene is generally regarded as strongly indicating its oncogenic role, although mutation rates of microsatellites are extremely variable, being influenced by flanking sequences, sequence composition, motif unit size, and recombination rate [26,36,37]. A bi-allelic inactivation and a role in a growth suppressor pathway are among other widely accepted criteria, but no consensus has been achieved, especially no mutation incidence cut-off value could be defined [18,48].…”

Flanking nucleotide specificity for DNA mismatch repair-deficient frameshifts within Activin Receptor 2 (ACVR2)

“…Plasmids pIREShyg2-ACVR2 exon 10-EGFP and pIREShyg2-ACVR2 exon 3-EGFP, containing portions of exon 10 and exon 3 of human ACVR2 were constructed previously [16, 17]. Plasmids pIREShyg2- ACVR2 exon 10-exon 3 flanking (F) OF and pIREShyg2- ACVR2 exon 3-exon 10 F OF were also constructed previously in which six nucleotides flanking ACVR2 exons 10 or 3 microsatellites at the 5’ and 3’ ends were swapped with the 6 nucleotides flanking ACVR2 exons 3 or 10 microsatellites, respectively [17].…”

“…We have measured frameshift mutation rate at the A 8 microsatellite of ACVR2 exon 10 in real time and showed that it accumulates −1 bp frameshift mutation in human colorectal cancer cells with DNA MMR defects (frameshifts with hMLH1 and hMSH6 deficiencies) after forming heteroduplexes at a constant rate [16, 17]. We also demonstrated frameshift mutation selectivity for the ACVR2 exon 10 over the ACVR2 exon 3 microsatellite in DNA MMR defective cells in real time [17]. Furthermore, we showed that this selectivity for frameshift mutation within ACVR2 lies in part with the immediate flanking nucleotides surrounding each coding microsatellite [17].…”

“…We also demonstrated frameshift mutation selectivity for the ACVR2 exon 10 over the ACVR2 exon 3 microsatellite in DNA MMR defective cells in real time [17]. Furthermore, we showed that this selectivity for frameshift mutation within ACVR2 lies in part with the immediate flanking nucleotides surrounding each coding microsatellite [17]. Briefly, we substituted a grouping of 6 nucleotides immediately the flanking 5’ and 3’ ends of each ACVR2 exon 3 and ACVR2 exon 10 microsatellite.…”

“…Briefly, we substituted a grouping of 6 nucleotides immediately the flanking 5’ and 3’ ends of each ACVR2 exon 3 and ACVR2 exon 10 microsatellite. Substitution of the flanking nucleotides surrounding the exon 10 microsatellite with those surrounding the exon 3 microsatellite greatly diminished heteroduplex (A 7 paired with T 8 ) and full mutant (A 7 paired with T 7 ) formation in hMLH1 −/− cells, while substitution of the flanking nucleotides from exon 3 with those from the exon 10 enhanced frameshift mutation [17]. These observations demonstrated a critical role of the flanking DNA sequence context for microsatellite frameshift mutation when DNA MMR is defective, possibly explaining why certain genes containing coding microsatellites become frameshift mutated at various frequencies.…”