Spontaneous and induced mutations at the human minisatellite MS32 in yeast

Abstract: Tandem repetitive DNA including minisatellites make up a large part of eukaryotic genomes, and some tandem repetitive loci are associated with human disease. Little is known about the functions and dynamics of these sequences. Hypervariable minisatellites are used as naturally occurring genetic markers and form the basis of DNA fingerprinting. Studies in human have shown that minisatellite alleles frequently mutate to new lengths by recombination-based mechanisms that operate in the germline, possibly in meiosis. In addition to the variability in length, all hypervariable minisatellites characterised to date also show variation in the DNA sequence of repeat units. The order of variant repeat units can be revealed by MVR-PCR (Minisatellite Variant Repeat mapping by PCR), and this has greatly contributed to mutation analysis by comparing structures of alleles before and after mutation. Certain aspects of minisatellite mutation and general eukaryotic meiotic recombination, cannot be studies in human or any other mammalian system. It was therefore necessary to develop a manipulable eukaryotic model system in the yeast Saccharomyces cerevisiae. The best characterised human minisatellite MS32 was integrated in the vicinity of a hotspot for meiotic recombination in chromosome III. This thesis presents the construction of the model system and analyses of MS32 mutation in yeast.The results proved that MS32 mutation is induced in meiosis. Mutant structures were strikingly similar to mutant structures seen in man. Tetrad analysis demonstrated that gene conversion is the major pathway leading to interallelic exchanges. The data also suggested that a hyper-recombinogenic state is formed, and it was shown that entire alleles can be transferred from a chromatid to another. An allele that displays reduced mutation rate in man showed a reduced mutation rate also in yeast. The results have implications for general eukaryotic meiotic recombination. Mutations at MS32 were induced in meiosis by PCB, suggesting that the model system can be used as an in vitro bioassay for the screening of environmental contaminants capable of inducing genomic damage in meiosis. It is concluded that the yeast model constitute a suitable system for the molecular dissection of pathways in spontaneous and induced minisatellite mutations and for elucidating general eukaryotic meiotic recombination mechanisms.