Deoxyribonucleic acid repair and genomic instability in yeast aging

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002.Includes bibliographical references.Aging is a universal process that occurs in eukaryotic organisms. Many features of aging, including the genetic pathways involved in aging, appear to be evolutionarily conserved. Extrachromosomal rDNA circles (ERCs) have been identified to be a cause of replicative aging in budding yeast S. cerevisiae. Genomic instability can cause ERCs to excise from chromosomal rDNA arrays, containing 35S and 5S rRNA genes. At each cell division, ERCs replicate via an origin of replication present in each rDNA repeat, accumulate asymmetrically in the mother cells due to their segregation bias, and ultimately cause nucleolar fragmentation and senescence. Introduction of an ERC into young cells shortens life span and accelerates the onset of age-associated sterility. ERCs are excised from the rDNA locus by homologous recombination. rad52 mutant cells, defective in DNA repair through homologous recombination, do not accumulate ERCs with age; likewise, mutations in other genes of the RAD52 class that have varying effects on homologous recombination have corresponding effects on ERC formation. rad52 mutation leads to a progressive delocalization of a silencing and DNA repair protein Sir3p from telomeres to other nuclear sites with age and, surprisingly, shortens life span despite the absence of ERCs. Spontaneous DNA damage, perhaps double-strand breaks (DSBs), are likely the cause of lethality in mutants of the RAD52 class and may be an initial step in aging in wild-type cells. Replication fork pausing in E. coli can cause DSBs. Consistently, afobl mutation, which abolishes unidirectional replication fork barrier (RFB) at the rDNA, reduces rDNA recombination, decreases ERC accumulation with age, and thus extends life span.(cont.) Therefore, reduction of ERC formation infobl cells is likely the consequence of an absence of DSBs caused by replication fork pausing at RFB, again suggesting that DSBs are the initial step in yeast aging. Foblp is also required for transcription enhancement from an ectopic RNA polymerase I (Pol I) promoter outside of the rDNA, but not at rDNA. The role of Foblp in this enhancement may be to facilitate interactions between rDNA repeats and the ectopic promoter, thus allowing recruitment of Pol I transcriptional machinery to the ectopic promoter. Consistent with this notion, Foblp influences the structural dynamics at rDNA. Foblp antagonizes Sir2p-dependent rDNA silencing and thus affects rDNA chromatin structure. fobl mutation partially suppresses the aberrant rDNA structure in topoisomerase I-deficient (topl) cells, likely by relieving torsional stress created during rDNA replication. In addition, afobl mutation was able to rescue the synthetic lethality in topl trf4 double mutant cells, due to the failure of mitotic condensation at rDNA. fobl mutation complemented several defects caused by trf4 mutation, suggesting that Trf4p, DNA polymerase [sigma], may have a specialized function at rDNA. FOB1 and likely RFB may play a direct role in the macromolecular rDNA structure that influences chromosome transmission and indirectly affects aging in yeast.by Peter Unnam Park.Ph.D