The target of rapamycin (TOR) pathway is an evolutionarily conserved nutrient sensing pathway that is repressed during caloric restriction, which is associated with extension of lifespan in yeast and higher eukaryotes. Replication stress, induced by inhibition of RNR and depletion of dNTP pools, shortens yeast chronological lifespan (CLS), which can be alleviated by glucose restriction or deletion of SCH9 (a downstream mediator of TOR signaling). To further investigate possible interaction between TOR signaling and replication stress, we used quantitative high throughput cell array phenotyping (Q-HTCP) of the complete collection of the yeast knockout and knockdown (YKO/KD) strains to identify genes that influence the TOR and RNR pathways. Based on this analysis and evidence from the literature, we hypothesize vacuolar acidification is a process where the TOR and RNR pathways functionally intersect, and suspect this gives rise to genetic interaction that could be important in determination of lifespan. There are several observations we are trying to relate with one another: Vacuolar acidification in glucose depleted media is optimal at pH = 7, and neutral pH is also associated with increased CLS, but whether increased vacuolar function affects, or is affected by, CLS is unclear. Interestingly, HU treatment can be associated with higher pH over time. We developed a high throughput pH assay to better associate pH and CLS across the deletion collection and CLS. Although hydroxyurea-treated yeast exhibit decreased viability initially, we found CLS to be paradoxically greater with increasing age, depending on MET17/met17-∆0 status. Deletion of MET17 extends CLS, yet it shortens late CLS after HU treatment whereas having a functional MET17 increases lifespan after HU treatment. Based on these findings, we are investigating a model whereby perturbation of RNR activity damages/kills some cells in S-phase, but may also induce inhibition of TOR signaling that facilitates G1/G0 arrest of surviving cells, which ultimately promotes extension of CLS.