ABSTRACT
Specific regions of genomes (fragile sites) are hot spots for the chromosome rearrangements that are associated with many types of cancer cells. Understanding the molecular mechanisms regulating the stability of chromosome fragile sites, therefore, has important implications in cancer biology. We previously identified two chromosome fragile sites in Saccharomyces cerevisiae that were induced in response to the reduced expression of Pol1p, the catalytic subunit of DNA polymerase alpha. In the study presented here, we show that reduced levels of Pol3p, the catalytic subunit of DNA polymerase delta, induce instability at these same sites and lead to the generation of a variety of chromosomal aberrations. These findings demonstrate that a change in the stoichiometry of replicative DNA polymerases results in recombinogenic DNA lesions, presumably double-strand DNA breaks.
Subject(s)
Chromosome Fragile Sites , Chromosome Fragility , DNA Polymerase III/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Base Sequence , Chromosomes, Fungal/metabolism , Crosses, Genetic , DNA Damage , Diploidy , Gene Deletion , Genes, Fungal , Haploidy , Mitosis/drug effects , Molecular Sequence Data , Mutation/genetics , Nucleic Acid Hybridization , Phenotype , Recombination, Genetic/genetics , Reproduction , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/geneticsABSTRACT
In the yeast Saccharomyces cerevisiae, reduced levels of the replicative alpha DNA polymerase result in greatly elevated frequencies of chromosome translocations and chromosome loss. We selected translocations in a small region of chromosome III and found that they involve homologous recombination events between yeast retrotransposons (Ty elements) on chromosome III and retrotransposons located on other chromosomes. One of the two preferred sites of these translocations on chromosome III involve two Ty elements arrayed head-to-head; disruption of this site substantially reduces the rate of translocations. We demonstrate that this pair of Ty elements constitutes a preferred site for double-strand DNA breaks when DNA replication is compromised, analogous to the fragile sites observed in mammalian chromosomes.
Subject(s)
Chromosomal Instability/genetics , Chromosome Fragile Sites/physiology , DNA Polymerase I/metabolism , Retroelements/genetics , Saccharomyces cerevisiae/genetics , Translocation, Genetic/genetics , Chromosome Fragile Sites/genetics , Chromosome Mapping , DNA Damage/genetics , DNA Polymerase I/genetics , DNA Repair/genetics , DNA Replication/genetics , Gene Expression Regulation, Fungal/genetics , Genome, Fungal , Oligonucleotide Array Sequence Analysis , Recombination, Genetic/genetics , Saccharomyces cerevisiae/enzymologyABSTRACT
Mismatch repair genes are important in maintaining the fidelity of DNA replication. To determine the function of the Caenorhabditis elegans homologue of the MSH2 mismatch repair gene (msh-2), we isolated a strain of C. elegans with an insertion of the transposable element Tc1 within msh-2. Early-passage msh-2 mutants were similar to wild-type worms with regard to lifespan and meiotic chromosome segregation but had slightly reduced fertility. The mutant worms had reduced DNA damage-induced germ-line apoptosis after genotoxic stress. The msh-2 mutants also had elevated levels of microsatellite instability and increased rates of reversion of the dominant unc-58(e665) mutation. In addition, serially passaged cultures of msh-2 worms died out much more quickly than those of wild-type worms. These results demonstrate that msh-2 function in C. elegans is important in regulating both short- and long-term genomic stability.
