Project description:We developed an artificial genome evolution system, which we termed ‘TAQing’, by introducing multiple genomic DNA double-strand breaks using a heat-activatable endonuclease in mitotic yeast. The heat-activated endonuclease, TaqI, induced random DSBs, which resulted in diverse types of chromosomal rearrangements including translocations. Array comparative genomic hybridization (aCGH) analysis was performed with cell-fused Saccharomyces cerevisiae strains induced genome evolution by TAQing system. Some of copy number variations (CNVs) induced by massive genome rearrangements were detected in the TAQed yeast strains.
Project description:Furfural is a key inhibitor in S. cerevisiae fermentation causing serious economic loss. To understand the toxic mechanisms of furfural-induced genomic instability and phenotypic evolution, we mapped chromosomal alterations in 21 furfural-treated yeast strains by whole genome SNP microarrays at a resolution about 1kb.
Project description:Yeast Saccharomyces cerevisiae has been widely used as a model system for studying genome instability. Here, heterozygous S. cerevisiae zygotes were generated to determine the genomic alterations induced by sudden introduction of active RNase H2. In combination of a custom SNP microarray, the patterns of chromosomal instability could be explored at a whole genome level. Ribonucleotides can be incorporated into DNA during replication by the replicative DNA polymerases. These aberrant DNA subunits are efficiently recognized and removed by Ribonucleotide Excision Repair, which is initiated by the heterotrimeric enzyme RNase H2. While RNase H2 is essential in higher eukaryotes, the yeast Saccharomyces cerevisiae can survive without RNase H2 enzyme, although the genome undergoes mutation, recombination and other genome instability events at an increased rate. Although RNase H2 can be considered as a protector of the genome from the deleterious events that can ensue from recognition and removal of embedded ribonucleotides, under conditions of high ribonucleotide incorporation and retention in the genome in a RNase H2-negative strain, sudden introduction of active RNase H2 causes massive DNA breaks and genome instability in a condition which we term “ribodysgenesis”. The DNA breaks and genome instability arise solely from RNase H2 cleavage directed to the ribonucleotide-containing genome. Survivors of ribodysgenesis have massive loss of heterozygosity events stemming from recombinogenic lesions on the ribonucleotide-containing DNA, with increases of over 1000X from wild-type. DNA breaks are produced over one to two divisions and subsequently cells adapt to RNase H2 and ribonucleotides in the genome and grow with normal levels of genome instability.
