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Human Molecular Genetics, 2002, Vol. 11, No. 6 707-713
© 2002 Oxford University Press

Sequence dependent instability of mononucleotide microsatellites in cultured mismatch repair proficient and deficient mammalian cells

Jayne C. Boyer1,+, Nazumi A. Yamada1, C. Natalia Roques1, Stephanie B. Hatch2, Kevin Riess1 and Rosann A. Farber1,2,3

1Department of Pathology and Laboratory Medicine, 2Curriculum in Genetics and Molecular Biology and 3Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

We have measured the mutation rates of G17 and A17 repeat sequences in cultured mammalian cells with and without mismatch repair and have compared these rates to those of a (CA)17 repeat sequence. Plasmids containing microsatellites that disrupt the reading frame of a downstream neomycin-resistance gene were introduced into the cells by transfection and revertants were selected using the neomycin analog G418. Comparison of mutation rates within cell lines showed that the mutation rates of A17 and (CA)17 sequences were similar in the mismatch repair proficient cells, but the mutation rate of G17 was significantly higher than that of either A17 or (CA)17. In the mismatch repair deficient cells, the G17 and (CA)17 mutation rates were similar and were significantly higher than the A17 rate. PCR analysis of the mutants showed that 1 bp insertions predominated in both mononucleotide repeats in the mismatch repair proficient cells; in mismatch repair deficient cells, 2 bp deletions were the most common mutation in the A17 sequence, but 1 bp insertions and 2 bp deletions were equally represented in the G17 sequence. These results indicate that a G17 repeat is less stable than an A17 repeat in both mismatch repair proficient and mismatch repair deficient mammalian cells. This observation implies that the replication fidelity is lower in G17 repeats.

+ To whom correspondence should be addressed. Tel: +1 919 966 6921; Fax: +1 919 966 0717; Email: jayne_boyer@med.unc.edu


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