Mechanisms of rDNA instability during aging

Project Summary. Genomic instability is one of the major hallmarks of aging conserved across all species. The repetitive ribosomal DNA (rDNA) locus is one of the most vulnerable genetic loci to various types of DNA damage, especially DNA double strand breaks (DSBs). The high level or rRNA transcription by RNA Polymerase I can conflict with the DNA replication forks to cause significant DNA replication stress. To prevent collisions, replication fork barriers (RFB) are typically established at the 3’ end of each active rDNA transcription unit. The classic eukaryotic example is the Fob1 protein in budding yeast Saccharomyces cerevisiae. Sloppy repair of Fob1-dependent RFBs trigger DSBs that can alter the rDNA copy number and produce extrachromosomal rDNA circles (ERCs) that are proposed to cause replicative aging. Fob1 has long been thought to function exclusively at the rDNA. However, we have recently identified several novel binding sites that also regulate rDNA stability and lifespan. Among these is the SIR2 gene, a major promoter of longevity through stabilization of the rDNA array. Our preliminary results implicate Fob1 in a regulatory network that functions with the Pol I transcription factor UAF to maintain rDNA copy number homeostasis through control of rDNA recombination via DSBs. This proposal further investigates the function of UAF and Fob1 in regulating DSBs and aging in yeast through an innovative whole genome DSB mapping technology that we will also apply to human IMR-90 cells to determine how the functionally analogous protein, transcription termination factor-1 (TTF1) controls RFB activity and DSB distribution in a replicative aging/senescence cell line model. To complement the functional investigation of Fob1 and TTF1, we will also determine the impact of aging on the 3D structure of Fob1 target sites via its role in recruiting the condensin complex. We anticipate the project will establish novel roles for Fob1 in regulating rDNA stability and aging in yeast and implicate TTF1 as a new regulator of rDNA stability in human cells.