Mutation rate and specificity analysis of tetranucleotide microsatellite DNA alleles in somatic human cells.
We have systematically varied microsatellite sequence composition to determine the effects of repeat unit size, G+C content, and DNA secondary structure on microsatellite stability in human cells. The microsatellites were inserted in frame within the 5' region of the herpes simplex virus thymidine kinase (HSV-tk) gene. The polypyrimidine/polypurine microsatellites displayed enhanced S1 nuclease sensitivity in vitro, consistent with the formation of non-B-form DNA structures. Microsatellite mutagenesis studies were performed with a shuttle vector system in which inactivating HSV-tk mutations are measured after replication in a nontumorigenic cell line. A significant increase in the HSV-tk mutation frequency per cell generation was observed after insertion of [TTCC/AAGG]9, [TTTC/AAAG]9, or [TCTA/AGAT]9 sequences (P <or= 0.0002), relative to the HSV-tk gene control. We observed that the G + C content of the microsatellite may affect mutagenesis, as the mean microsatellite mutation rates of the [TTTC/AAAG]9 and [TCTA/AGAT]9 alleles were sevenfold and 11-fold higher, respectively, than the [TTCC/AAGG]9 allele. A bias toward expansion mutations was noted for the majority of clones bearing the [TTCC/AAGG]9 allele as well as a [TC/AG]17 microsatellite of similar allele length. The mean microsatellite mutation rate of the [TTCC/AAGG]9 allele did not differ significantly from that for a [TC/AG]11 allele, demonstrating that these tetranucleotide and dinucleotide alleles are of equivalent stability. It is known that microsatellite mutagenesis is affected by the number of repeat units within an allele. Our data suggest that additional biochemical factors may regulate both the rate and specificity of somatic cell microsatellite mutagenesis.
Jake Gittlen Cancer Research Institute, Department of Pathology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033, USA.
Kristin A
KA
Eckert
Guang
G
Yan
Suzanne E
SE
Hile
D000483:Alleles; D002478:Cells, Cultured; D004252:DNA Mutational Analysis / Q000379:methods; D005260:Female; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D018895:Microsatellite Repeats; D016296:Mutagenesis; D009154:Mutation; D018139:Simplexvirus / Q000235:genetics; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000235:genetics / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics
D013937:Thymidine Kinase; D015719:Single-Strand Specific DNA and RNA Endonucleases