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.