N. Alice S. Yamada, Ph.D.

Dissertation research performed under the direction of Rosann A. Farber

ABSTRACT
    Microsatellites are repetitive DNA tracts consisting of tandem arrays of one to five base pairs, with five to thirty repeat units per tract.  Microsatellites are particularly prone to frameshift mutations via insertion-deletion loop formation during DNA synthesis.  The mismatch repair pathway corrects most of these replication errors; microsatellite mutation rates are significantly elevated in the absence of mismatch repair.  The research presented in this dissertation explored whether factors other than mismatch repair defects can also cause elevation of microsatellite mutation rates in telomerase-immortalized normal human fibroblasts (hTERT-1604).  Cell lines were developed from hTERT-1604 cells by stable transfection of plasmids containing a (CA)17, A17, or G17 repeat.  The microsatellites were inserted upstream of a bacterial neomycin-resistance gene (neo) in the plasmid, such that the coding region of the neo gene was placed out of frame.  Revertants with frameshift mutations in the microsatellite were selected by growth of the cells in G418.  Fluctuation tests were carried out to measure mutation rates under various conditions.  The results indicate that 1) there is a ten-fold difference in microsatellite mutation rates between two cancer cell lines with null mutations in different mismatch repair genes, suggesting the possibility that inactivation of various mismatch repair genes may not lead to equal levels of microsatellite instability, although other properties of these cell lines may also contribute to these differences; 2) overexpression of an error-prone polymerase, pol b, brings about an elevation in microsatellite mutation rates; 3) oxidative DNA damage induces microsatellite mutations in dinucleotide, but not mononucleotide, repeats; and 4) replication errors that lead to two-bp insertions in short microsatellites and large deletions (>8 bp) in long microsatellites may be repaired with lower efficiency than those that result in other frameshift mutations.  This study identified novel mechanisms for the induction of microsatellite instability and provided evidence that low levels of microsatellite instability can occur in the presence of mismatch repair activity.  These results may explain why modest microsatellite instability is observed in some tumor cells with no known mismatch repair defects.