Mutation rate of a microsatellite sequence in normal human fibroblasts

Area of Science:

• Genetics
• Molecular Biology
• Genomics

Background:

• Dinucleotide repeats are susceptible to frameshift mutations, often through DNA polymerase slippage.
• Microsatellite DNA sequences exhibit high mutation rates in mismatch repair-defective cells.
• Previous studies in normal cells were limited to maximal mutation rate estimates due to low assay sensitivity.

Purpose of the Study:

• To directly measure the spontaneous mutation rate of a dinucleotide repeat in diploid human cells.
• To establish a sensitive system for detecting microsatellite mutations in normal cells.
• To provide a precise quantification of microsatellite instability in the absence of DNA repair defects.

Main Methods:

• Utilized a stably integrated plasmid containing a (CA)17 repeat in human foreskin fibroblasts.
• The dinucleotide repeat was engineered to disrupt the reading frame of a neomycin resistance gene.
• Cells with frameshift mutations restoring neomycin gene function were selected using G418, enabling sensitive mutation detection.

Main Results:

• The developed system allowed for highly sensitive measurement of microsatellite mutation rates.
• Fluctuation analysis yielded spontaneous mutation rates ranging from less than 3.1 x 10^-8 to 44.8 x 10^-8 mutations per cell per generation.
• This represents the first direct measurement of a spontaneous microsatellite mutation rate in normal human cells.

Conclusions:

• The study successfully quantified the spontaneous mutation rate of microsatellites in normal human cells.
• The findings provide crucial baseline data for understanding DNA instability in healthy cells.
• This sensitive assay system opens avenues for further research into factors influencing microsatellite stability.