title: 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 (A(7)/T(8)) and full (A(7)/T(7)) 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 +1bp out-of-frame of EGFP, allowing a -1bp 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.
authors: Department of Medicine, University of California, San Diego, CA, United States.
authors: Heekyung
authors: H
authors: Chung
authors: Joy
authors: J
authors: Chaudhry
authors: Jenny F
authors: JF
authors: Lai
authors: Dennis J
authors: DJ
authors: Young
authors: John M
authors: JM
authors: Carethers
mesh_terms: D030301:Activin Receptors, Type II / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D053843:DNA Mismatch Repair; D049914:DNA Repair-Deficiency Disorders / Q000235:genetics / Q000378:metabolism; D005091:Exons; D005434:Flow Cytometry / Q000379:methods; D016368:Frameshift Mutation; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics; D008969:Molecular Sequence Data; D059645:Mutation Rate; D009711:Nucleotides / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D015398:Signal Transduction
chemical_list: D009711:Nucleotides; D018125:Receptors, Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D030301:Activin Receptors, Type II; D000077294:Receptor, Transforming Growth Factor-beta Type II; C425585:activin receptor type II-A