SPONTANEOUS MUTAGENESIS OF TRIPLET REPEATS

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Within the last three years the molecular basis of seven human genetic diseases; including Fragile X-syndrome (FRAXA and FRAXE), myotonic dystrophy, Kennedy's disease, Huntington's disease, spinocerebellar ataxia type 1 (SCA1), and dentatorubral pallidoluysian atrophy, has been described. These hereditary diseases are similar in that they show genetic anticipation, in which the severity of the disease increases and the age of onset decreases in successive generations. The molecular basis for anticipation appears to be the expansion of tracts of triplet repeats (CGG in the case of FRAXA and FRAXE and CTG in the other five diseases). In Fragile X-syndrome and myotonic dystrophy, the length of triplet repeat (usually less than 40 repeats in normal individuals) can expand to thousands of repeats in severely affected individuals. Size heterogeneity and especially deletion of repeats in DNA are common and presumably due to slipped misalignment during DNA replication or recombination. The remarkable tendency of triplet repeats in humans to expand represents a novel genetic phenomenon, which has not been previously described. This project will determine if replication and/or recombination-based errors are responsible for this unusual genetic phenomena. Using triplet repeat sequences containing interruptions from normal Fragile X (CGG repeats) and SCA1 individuals (CTG repeats) as well as long uninterrupted repeats, we will investigate the effect of repeat sequence and length on DNA polymerase induced slippage and/or strand displacement. We will test the hypothesis that a block to replication at triplet repeats induces reiterative synthesis resulting in expansion. Replication of repeats will be studied in Xenopus eggs and egg extracts, a system that mimics replication in early development (where expansion in some diseases is thought to occur). The genetic stability of triplet repeats will be determine in E. coli, yeast, Chinese hamster ovary (CHO), mouse embryonic stem (ES), and human cells in wild type or normal) cells and in cells containing mutations affecting DNA replication, mismatch repair, and genetic recombination. Selection in yeast will employ ura3. In CHO, ES, and human cells selection will utilize exon skipping int eh APRT or HPRT gene. Genetically modified ES cells, with triplet repeats in the second intron or in an artificial third exon in the HPRT gene will be used to measure the frequency of recombination events associated with the repeats. ES cells and transgenics deficient in rep3, a MutS mismatch repair homologue, will be made and tested for triplet repeat stability. The effects of environmental mutagens, carcinogens, and chemicals on the fidelity of DNA replication of repeats will be investigated.