Somatic mutation rates and specificities at TC/AG and GT/CA microsatellite sequences in nontumorigenic human lymphoblastoid cells.
We have examined mutational events at TC/AG microsatellites, the second most abundant dinucleotide repetitive motif in the human genome. Mutational targets were constructed containing TC/AG alleles up to 20 units in-frame within the coding region of the herpes simplex virus thymidine kinase (HSV-tk) gene. These targets were incorporated into oriP shuttle vectors, which replicate episomally in human lymphoblastoid cells. The overall HSV-tk mutant frequencies measured after 10 population doublings in cells derived from a clinically normal donor were slightly increased over the background of mutations recovered in Escherichia coli. DNA sequence analyses revealed that replication of TC/AG vectors in human cells increased the mutation frequencies at the microsatellite motif up to 3-fold, relative to background. Additionally, the median HSV-tk mutation rate of single-cell clones carrying the [TC/AG]17 vector was significantly different from that of clones harboring the control vector. The median rate of allele length alterations within the [TC/AG]11 tract was 2 x 10(-6) mutations/cell generation, with an equivalent rate of deletion and expansion mutations. In contrast, a [GT/CA]10 vector showed no increase in microsatellite mutation frequency after replication in human cells, and mutation rates of clones carrying a [GT/CA]16 vector were not significantly different from controls. Intriguingly, replication in human cells of all microsatellite-containing vectors resulted in elevated mutation frequencies at the downstream HSV-tk coding sequence of up to 20-fold, an effect not observed for the control vector. These results demonstrate that the frequency of mutational events at TC/AG motifs is slightly greater than at GT/CA motifs of similar allele length. This is the first report to our knowledge of the mutation rates at TC/AG microsatellite alleles in eukaryotic or prokaryotic cells.
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The Jake Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine, Hershey 17033, USA.
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D001483:Base Sequence; D002461:Cell Line, Transformed; D004261:DNA Replication; D004274:DNA, Recombinant / Q000235:genetics; D018900:Dinucleotide Repeats / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005787:Gene Frequency; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism*; D018895:Microsatellite Repeats / Q000235:genetics*; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase