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

Abstract

We previously demonstrated that exonic selectivity for frameshift mutation (exon 10 over exon 3) of ACVR2 in mismatch repair (MMR)-deficient cells is partially determined by 6 nucleotides flanking 5’ and 3’ of each microsatellite. Substitution of flanking nucleotides surrounding the exon 10 microsatellite with those surrounding the exon 3 microsatellite greatly diminished heteroduplex (A7/T8) and full (A7/T7) mutation, while substitution of flanking nucleotides from exon 3 with those from exon 10 enhanced frameshift mutation. We hypothesized that specific individual nucleotide(s) within these flanking sequences control ACVR2 frameshift mutation rates. Only the 3rd nucleotide 5’ of the microsatellite, and 3rd, 4th, and 5th nucleotides 3’ of the microsatellite were altered from the native flanking sequences and these locations were individually altered (sites A, B, C, and D, respectively). Constructs were cloned +1 bp out-of-frame of EGFP, allowing a −1 bp frameshift to express EGFP. Plasmids were stably-transfected into MMR-deficient cells. Non-fluorescent cells were sorted, cultured for 35 days, and harvested for flow cytometry and DNA-sequencing. Site A (C to T) and B (G to C) in ACVR2 exon 10 decreased both heteroduplex and full mutant as much as the construct containing all 4 alterations. For ACVR2 exon 3, site A (T to C), C (A to G), and D (G to C) are responsible for increased heteroduplex formation, whereas site D is responsible for full mutant formation by ACVR2 exon 10 flanking sequences. Exonic selectivity for frameshift mutation within ACVR2’s sequence context is specifically controlled by individual nucleotides flanking each microsatellite.

Oxidative Stress Induces Nuclear-to-Cytosol Shift of hMSH3, a Potential Mechanism for EMAST in Colorectal Cancer Cells

“…Individual sequence context and flanking sequences may also contribute to the different outcome of the mutation (e.g. insertion vs. deletion) [11], [12], [24]. At the D20S82 locus, only about one-third of EGFP-positive hMSH3 −/− cells demonstrated one AAAG unit deletion and this could be due to this particular clone containing multiple copies of the construct.…”

“…Sense and anti-sense oligos containing D8S321 or D20S82 , surrounding sequences, as well as Pme I and Asc I cloning sites ( Table S1 ) were synthesized (Integrated DNA Technologies, Inc., San Diego, CA) and subjected to T4-polynucleotide kinase reactions to phosphorylate the 5′ ends (Invitrogen). The sense and anti-sense oligos were allowed to anneal and cloned into pIREShyg2-EGFP plasmids via Pme I- Asc I sites immediately after the start codon of the EGFP gene as described previously [10]–[12], [24]. Experimental plasmids were constructed +1 bp out of frame, such that one deletion of a tetranucleotide repeat within D8S321 or D20S82 (−4 bp frameshift mutation) would shift the reading frame into the correct frame to allow EGFP expression.…”

“…Mutation rates were calculated by the “method of the mean” developed by Luria and Delbruck [25] as described previously [10]–[12], [24].…”

Microsatellite Instability Pathway and EMAST in Colorectal Cancer

“…This fact is believed to be the driver of improved outcome for patients with MSI-H CRCs. The frameshifted genes are transcribed and translated as shorter novel peptides due to the frameshift and new stop codon (Figure 2) [1–3,40–45]. These neopeptides are immunogenic and are recognized by the immune system, which causes the development of subepithelial lymphoid aggregates.…”

Tetranucleotide Microsatellite Mutational Behavior Assessed in Real Time: Implications for Future Microsatellite Panels

“…5 We and others previously have shown that mononucleotide and dinucleotide microsatellites in the absence of MMR consistently and uniformly frameshift to shorter microsatellite lengths. [35][36][37][38][39][40] For this to occur, the insertion/deletion loop needs to occur on the template DNA strand, allowing the newly synthesized DNA strand to anneal a shortened complementary microsatellite sequence. [35][36][37][38][39] In the absence of MLH1 or with knockdown of MSH3, we initially observed both deletions and insertion length changes at tetranucleotide sequences, suggesting that insertion/deletion loops form at these longer microsatellites on both the template and newly synthesized DNA strand.…”