Within the last three years mutations responsible for 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, have been described. Several of these hereditary diseases show anticipation, in which the severity of the disease increases and/or the age of onset decreases in successive generations. The mutations found in each of these disorders involve increased lengths of trinucleotide repeats (CGG and CTG) which are genetically unstable. The instability of the repeats likely accounts for the genetic anticipation observed. Expanded repeats lengths are found in more severely affected patients; thus it is important to understand the molecular mechanisms involved in repeat expansion. This Program Project Grant will test several hypothesis for the expansion of triplet repeats. Program I presents preliminary investigations demonstrating that (CTG)n triplet repeats adopt non-B DNA structures and that slippage at repeats occurs in E. coli. Structures formed by all ten trinucleotide repeats will be characterized, and their involvement in replication pausing and recombination will be investigated. Program II will investigate potential slipped mis-paired structures that are likely to form in these sequences. Proteins that bind specifically to triplet repeats will be characterized. The effect of expanded triplet repeats on chromatin structure and gene expression will be studied. Program III will assay expansion during replication in vitro and in vivo (including Xenopus eggs and extracts), and test the hypothesis that a replication block at repeats induces reiterative (expansive) synthesis. The stability of triplet repeats in E. coli, yeast, CHO, mouse embryonic stem (ES), and human cells will be studied. The role of genetic recombination in repeat instability will be examined in normal and mismatch repair deficient (rep3) ES cells. Program IV will utilize human DNA polymerases and test the hypothesis that primer relocation leads to expansion. This project will also investigate replication in extracts of human cells from individuals affected with anticipation-associated diseases, as well in extracts from cells deficient in DNA repair, mismatch repair, or DNA replication. Program V will develop a model system utilizing ES cells for examining the stability of triplet repeats in sequences from normal and affected individuals to determine if the instability is inherent for the repeat alone. Moreover, this project will provide biologically relevant materials to the project, including sequences from normal and affected individuals. This group of experienced investigators with proven records of accomplishments blends substantial expertise to investigate a problem in molecular medicine of great genetic and clinical importance. This highly focused project will fill a void by providing new information on the molecular basis for mutational events underlying several important human diseases.
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