Flanking sequence specificity determines coding microsatellite heteroduplex and mutation rates with defective DNA mismatch repair (MMR).
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 (A(7)/T(8)) in hMLH1(-/-) cells, but only exon 10 microsatellites fully mutated (A(7)/T(7)) 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.
Department of Medicine, University of California, San Diego, CA, USA.
H
H
Chung
C G
CG
Lopez
D J
DJ
Young
J F
JF
Lai
J
J
Holmstrom
D
D
Ream-Robinson
B L
BL
Cabrera
J M
JM
Carethers
D030301:Activin Receptors, Type II / Q000235:genetics; D048868:Adaptor Proteins, Signal Transducing / Q000172:deficiency; D020137:Base Pair Mismatch / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D004260:DNA Repair / Q000235:genetics*; D021901:DNA, Intergenic / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D000070957:MutL Protein Homolog 1; D016296:Mutagenesis / Q000235:genetics*; D009687:Nuclear Proteins / Q000172:deficiency
D048868:Adaptor Proteins, Signal Transducing; D021901:DNA, Intergenic; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D030301:Activin Receptors, Type II; D000070957:MutL Protein Homolog 1