Recombination mediated instabilities of (CTG:CAG) and (CAG:CTG) triplet repeat sequences from human hereditary disease

The number of human hereditary diseases associated with the expansion of triplet repeat sequences continues to increase over the years (1). Since 1991 with the identification of fragile X syndrome as a trinucleotide disease, 15 different human hereditary diseases have been shown to share a common phenomenon of length polymorphisms of triplet repeat sequences (TRS) (2). The change in size of the repeat sequence is dramatic in some cases, while in other cases only small increments occur. We propose that gene conversion (recombination) events mediate expansions of CTG•CAG repeat sequences. Although there were clinical reports of recombination mediated instability of TRS, the role recombination played in genetic instability of TRS remained uninvestigated.;To gain insights into the effects of recombination on triplet repeat instability, we designed a two plasmid recombination system. Our experiments involved the use of E. coli as a model system to investigate the effects of various parameters on recombination mediated instability: Such as the type of triplet repeat sequence, the length of the triplet repeat sequence, presence of point mutations, sequence orientation and the determination of whether gene conversion or crossing-over is a pathway by which expansions occur.;This thesis shows that recombination mediates expansions of CTG•CAG repeat sequences in E. coli. Presumably, this is due to the generation of double strand breaks within the repeat. The length polymorphism was independent of tract orientation, but dependent on the absence of point mutations.;Many different types of sequences were investigated in our two plasmid recombination system. The sequences CTG•CAG and CAG•CTG had the greatest propensity for expansions of length. There was a threshold for recombination mediated instability at 30 repeats. However, when long tracts of CAG•CTG, which are above the threshold length for instability, had G - to - A point mutations within the repeat tract there were little to no expansion products. Thus, as low as 2.1% heterology is sufficient to disrupt the process of recombination mediated expansions. Finally, extensive restriction mapping and DNA sequencing of the expansion products confirmed that gene conversion was the pathway by which recombination mediated the length alterations.