SIRT1 Prevents R-Loops during Chronological Aging by Modulating DNA Replication at rDNA Loci

Abstract

In metazoans, the largest sirtuin, SIRT1, is a nuclear protein implicated in epigenetic modifications, circadian signaling, DNA recombination, replication, and repair. Our previous studies have demonstrated that SIRT1 binds replication origins and inhibits replication initiation from a group of potential initiation sites (dormant origins). We studied the effects of aging and SIRT1 activity on replication origin usage and the incidence of transcription–replication collisions (creating R-loop structures) in adult human cells obtained at different time points during chronological aging and in cancer cells. In primary, untransformed cells, SIRT1 activity declined and the prevalence of R-loops rose with chronological aging. Both the reduction in SIRT1 activity and the increased abundance of R-loops were also observed during the passage of primary cells in culture. All cells, regardless of donor age or transformation status, reacted to the short-term, acute chemical inhibition of SIRT1 with the activation of excessive replication initiation events coincident with an increased prevalence of R-loops. However, cancer cells activated dormant replication origins, genome-wide, during long-term proliferation with mutated or depleted SIRT1, whereas, in primary cells, the aging-associated SIRT1-mediated activation of dormant origins was restricted to rDNA loci. These observations suggest that chronological aging and the associated decline in SIRT1 activity relax the regulatory networks that protect cells against excess replication and that the mechanisms protecting from replication–transcription collisions at the rDNA loci manifest as differentially enhanced sensitivities to SIRT1 decline and chronological aging.